Patch graft compositions for cell engraftment

ABSTRACT

Compositions and methods of transplanting cells by grafting strategies into solid organs (especially internal organs) are provided. These methods and compositions can be used to repair diseased organs or to establish models of disease states in experimental hosts. The method involves attachment onto the surface of a tissue or organ, a patch graft, containing the donor cells. The donor cells may be a mixture of stem cells/progenitors with supporting early lineage stage mesenchymal cells. The patch graft promotes migration of the donor cells into the host organ and supports the successful integration of donor cells with host cells to repair the diseased organ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/US2018/036960, filed Jun. 11, 2018, which claimspriority from U.S. Provisional Patent Application Nos. 62/518,380, filedJun. 12, 2017, and 62/664,694, filed Apr. 30, 2018. The contents ofthese applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention is directed generally to the field oftransplantation of cells or tissue engrafting. More specifically, fromsolid organs into solid organs, especially to internal organs. Theinvention concerns compositions and methods providing strategies for therapid transplantation, engraftment and integration of cells into tissuesof solid organs to treat diseases of solid organs or to establish modelsystems of a disease. Representative of this potential are celltherapies for treatment of hepatic or pancreatic diseases.

BACKGROUND

There is an unmet need for grafting strategies involving cells derivedfrom solid organs, strategies distinct from those used fortransplantation of hemopoietic cells, mesenchymal stem cells or forskin. Transplantation of hemopoietic cells and mesenchymal cells,usually from single cell suspensions, is done routinely via a vascularchannel and is dependent on activation of adhesion molecules in relevanttarget sites because of micro-environmental signaling, a processreferred to as “homing”. Methods used for skin employ grafting methodswith cells and/or tissues applied directly to target sites.Transplantation of cells from solid organs other than skin have beendelivered traditionally via a vascular route or injected directly intothe tissue. This approach is not logical, since adhesion molecules oncells from solid organs are always activated and result in rapid(seconds) cell aggregation that can generate life-threatening emboli.Even if emboli are managed successfully to minimize health risks, theefficiency of cell engraftment is low, only ˜20% or less for adult cellsand even lower (<5%) for stem/progenitors. Most transplanted cellseither die or are transported to ectopic sites, where they can live formonths, creating tissue in inappropriate sites resulting in possibleadverse effects clinically. The small percentage of cells that engraftinto target sites slowly integrate, requiring weeks to months toreconstitute a significant portion of the tissue.

There is improvement in engraftment in liver and minimizing of ectopiccell delivery if cells are coated with hyaluronans and delivered via avascular route due to the tissue's (e.g. liver's) clearance ofhyaluronans. However, this improvement is still less efficient than thatwith grafting strategies and, importantly, still allows for some (albeitless) delivery of cells to ectopic sites.

Grafts for solid organs are challenging to design because of concernswith respect to size, shape, and complexity in the structure of organsin addition to the dynamic mechanical forces. Accordingly, there remainsa need for improved methods of cell engraftment into solid organs. Thisdisclosure fulfills this need and provides related advantages.

SUMMARY

Described herein are novel patch graft compositions and methods fortransplantation of cells into tissues and solid organs.

In one aspect, the present disclosure relates to a method of engraftingcells into a solid organ of a subject in need thereof, comprising:

-   -   contacting a patch graft onto a solid organ,        -   the patch comprising a mixture of epithelial cells and            mesenchymal cells incorporated into a biomaterial having a            first viscoelasticity property, in which the biomaterial            promotes an engraftment of at least a portion of said            epithelial cells, mesenchymal cells, or both among the cells            of the solid organ;    -   demonstrating that at least a portion of said epithelial cells,        mesenchymal cells, or both have engrafted among the cells of the        solid organ.

In some embodiments, demonstrating comprises measuring a level of asecretion from the solid organ, or a metabolic effect of the solidorgan, in a biological sample obtained from the subject to demonstratethat at least a portion of said epithelial cells have engrafted amongthe cells of the solid organ.

In another aspect, the present disclosure relates to a method ofengrafting cells into a solid organ of a subject in need thereof,comprising:

-   -   contacting a patch graft onto a solid organ,        -   the patch comprising a mixture of epithelial cells and            mesenchymal cells incorporated into a hydrogel layer having            a first viscoelasticity property, in which the hydrogel            promotes a migration of at least a portion of said            epithelial cells, mesenchymal cells, or both from the patch            through an outer surface of the solid organ.    -   demonstrating that at least a portion of said epithelial cells,        mesenchymal cells, or both have migrated through an outer        surface of the solid organ.

In some embodiments, demonstrating comprises measuring a parameter or achange in same, which indicates a physiological effect in the subjectresulting from the migrated cells.

In some embodiments, the patch graft further comprises a backing thatpromotes a migration of at least a portion of the mixture of epithelialcells and mesenchymal cells towards the solid organ.

In some embodiments, at least a portion of the mixture of epithelialcells and mesenchymal cells migrates over the substantial width of thesolid organ and distributes throughout the solid organ.

In some embodiments, the solid organ is an endodermal organ.

In some embodiments, the solid organ is an endodermal organ comprisingliver, pancreas, intestine, lung, bile duct, thymus, thyroid,parathyroid and the urogenital sinus region of the prostate and vagina.

In some embodiments, the endodermal organ comprises liver, andengraftment involves a remodeling of Glisson's Capsules.

In some embodiments, the present disclosure provides methods whichfurther gives rise to a combination of (i) engrafted epithelial cellsand mesenchymal cells and (ii) host cells.

In some embodiments, the methods of the present disclosure gives rise tofunctional hepatic parenchymal cells.

In some embodiments, the parenchymal cells comprise hepatocytes andcholangiocytes.

In some embodiments, the endodermal organ comprises pancreas, andengraftment involves a remodeling of pancreatic capsules and pancreatictissue near to the graft site.

In some embodiments, the methods of the present disclosure gives rise tofunctional pancreatic cells.

In some embodiments, the functional pancreatic cells comprise acinarcells and islets.

In some embodiments, the pancreas secretes increased levels of at leastone of insulin, c-peptide, glucagon, somatostatin, or pancreaticpolypeptide.

In some embodiments, the pancreas exhibits a metabolic effect of reducedblood sugar levels.

In some embodiments, the pancreas exhibits a metabolic effect ofincreased glucose tolerance.

In some embodiments, the pancreas secretes increased levels of adigestive enzyme or bicarbonate fluid.

In some embodiments, the digestive enzyme comprises amylase, lipase,peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, orcombinations thereof.

In some embodiments, the methods of the present disclosure results inincreased levels of a metabolic product derived from a digestive enzymesecreted by the pancreas.

In some embodiments, the digestive enzyme comprises amylase, lipase,peptidase, ribonuclease, deoxyribonuclease, gelatinase, elastase, orcombinations thereof.

In some embodiments, the methods of the present disclosure results inimproved digestion.

In some embodiments, the liver secretes urea, bile acids, phospholipids,lipoproteins, bilirubin, bicarbonate-rich fluids, or blood-clottingfactors.

In some embodiments, a biological sample obtained from the subjectindicates reduced levels of at least one of cholesterol, blood sugar,alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactatedehydrogenase.

In some embodiments, the patch includes a backing positioned over thehydrogel containing of the mixture of epithelial cells and mesenchymalcells.

In some embodiments, the backing is used to tether the hydrogel layer tothe host organ.

In some embodiments, at least one of the epithelial cells, mesenchymalcells, or both are early lineage stage cells.

In some embodiments, the early lineage stage mesenchymal cells (ELSMCs)comprise angioblasts, precursors of endothelia, stellate cells, orcombinations thereof.

In some embodiments, the early lineage stage epithelial cells (ELSEs),ELSMCs, or both are derived from embryonic stem (ES) cells or frominduced pluripotent stem cells (iPS).

In some embodiments, the epithelial cells are mature and the mesenchymalcells are ELSMCs.

In another aspect, the present disclosure relates to a method ofintroducing, restoring, increasing, or improving functionality of adiseased, impaired, or malfunctioning solid organ of a subject,comprising contacting the diseased, impaired, or malfunctioning solidorgan with a patch graft comprising a mixture of epithelial cells andmesenchymal cells under conditions that promote engraftment of theepithelial cells and mesenchymal cells; demonstrating an introduction,restoration, increase, or improvement of a functionality in thediseased, impaired, or malfunctioning solid organ.

In some embodiments, demonstrating comprises measuring in a biologicalsample obtained from the subject a level of a secretion or metabolicproduct or effect.

In some embodiments, the methods of the present disclosure furthercomprises demonstrating that a least a portion of the mixture ofepithelial cells and mesenchymal cells has distributed among the cellsof the host organ.

In some embodiments, an exposed surface of the patch graft includes acoating that inhibits adhesion of the patch graft to organs and tissuesin the vicinity of the patch graft.

In some embodiments, the solid organ comprises an endodermal organ.

In some embodiments, the endodermal organ comprises liver, pancreas,intestine, lung, bile duct, thymus, thyroid, parathyroid or the regionsfrom the urogenital sinus of the prostate or vagina.

In some embodiments, the solid organ comprises a pancreas and in whichan increased level of the secretion of at least one of insulin,c-peptide glucagon, somatostatin, or pancreatic polypeptide is measured.

In some embodiments the solid organ comprises a pancreas and in which areduced blood sugar level is measured.

In some embodiments, the solid organ comprises a pancreas and in whichincreased glucose tolerance is demonstrated.

In some embodiments, the solid organ comprises a pancreas and in whichincreased levels of a digestive enzyme or bicarbonate fluid isdemonstrated.

In some embodiments, the digestive enzyme comprises amylase, lipase,peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase.

In some embodiments, the solid organ comprises a pancreas and in whichincreased levels of a product from a digestive enzyme secreted by thepancreas is measured.

In some embodiments, the digestive enzyme comprises amylase, lipase,peptidase, ribonuclease, deoxyribonuclease, gelatinase, or elastase.

In some embodiments, the solid organ comprises a pancreas and in whichimproved digestion is demonstrated.

In some embodiments, the solid organ comprises liver, and in which asecretion comprises urea, bile acids, phospholipids, lipoproteins,bilirubin, bicarbonate-rich fluids, blood-clotting factors, orcombinations thereof.

In some embodiments, the solid organ comprises liver, and in which ametabolic effect is a reduced level of one or more of cholesterol, bloodsugar, alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactatedehydrogenase.

In some embodiments, the solid organ comprises a liver, and the subjectsuffers from type 1 tyrosinemia.

In some embodiments, a metabolic effect is a decrease in levels oftyrosine or alpha-fetoprotein.

In another aspect, the present disclosure relates to a method oftreating a subject diagnosed with a pathological condition attributableat least in part to having a diseased, impaired, or malfunctioning solidorgan, comprising

-   -   (i) contacting the diseased, impaired, or malfunctioning solid        organ with a patch graft comprising a mixture of epithelial        cells and mesenchymal cells.    -   (ii) allowing the epithelial cells and mesenchymal cells to        migrate into and distribute among the cells of the host solid        organ, and    -   (iii) demonstrating that a negative effect of said diseased,        impaired, or malfunctioning solid organ has been alleviated in        the treated subject.

In some embodiments, demonstrating comprises measuring in a biologicalsample obtained from the subject a level of a secretion or a metabolicproduct or effect.

In some embodiments, the migration and distribution steps lead to analleviation of the disease, impairment, or malfunction.

In some embodiments, the solid organ is an endodermal organ.

In some embodiments, the endodermal organ comprises liver, pancreas,intestine, lung, bile duct, thymus, thyroid, parathyroid, and theurogenital sinus regions of the prostate or vagina.

In some embodiments, the endodermal organ is pancreas and in which thesubject suffers from diabetes.

In some embodiments, increased levels of at least one of insulin,c-pcptide, glucagon, somatostatin, or pancreatic polypeptide ismeasured.

In some embodiments, reduced blood sugar levels are demonstrated.

In some embodiments, increased glucose tolerance is demonstrated.

In some embodiments, the subject comprises a mammal.

In some embodiments, the mammal is human.

In another aspect, the present disclosure relates to a patch graftcomprising a mixture of epithelial cells and mesenchymal cells and oneor more biomaterial layers including, at least:

-   -   a) a first, inner layer for contacting a solid organ, the first        inner layer exhibiting a first viscoelastic property,        incorporating a mixture of epithelial cells and mesenchymal        cells, supporting an ability of the epithelial cells and the        mesenchymal cells to produce secreted matrix metallo-proteinases        (MMPs) and promoting viability and immaturity of said epithelial        cells and mesenchymal cells;    -   b) optionally, a backing that confers a barrier to the cells        migrating in a direction other than towards the solid organ, the        backing exhibiting a second viscoelastic property; and    -   c) optionally, a third, outer layer of a coating or material        that minimizes adhesions of the patch graft to internal surfaces        of a body cavity, including internal walls and/or organs, in        proximity to the patch graft; wherein said viscoelastic        properties are determined by measuring rheological traits and        expressed in Pascals (Pa).

In some embodiments, the epithelial cells comprise early lineage stageepithelia cells (ELSEs) and the mesenchymal cells comprise early lineagestage mesenchymal cells (ELSMCs), or in which the epithelial andmesenchymal cells are of later lineage stages but are of comparablelineage stages as each other.

In some embodiments, the ELSMCs comprises angioblasts, precursors ofendothelia, stellate cells, or combinations thereof.

In some embodiments, the ELSEs and/or the ELSMCs are derived fromembryonic stem (ES) cells or from induced pluripotent stem cells (iPS).

In some embodiments, the epithelial cells are of a later lineage stageand the mesenchymal cells are early lineage stage mesenchymal cells(ELSMCs), or the mesenchymal cells are of a later lineage stage and theepithelial cells are early lineage stage epithelial cells (ELSEs).

In some embodiments, the second viscoelastic property (expressed in Pa)has a greater value than the first viscoelastic property.

In some embodiments, the one or more biomaterial layers comprise ahydrogel, which further comprises minimally sulfated or non-sulfatedglycosaminoglycans.

In some embodiments, the non-sulfated glycosaminoglycans comprisehyaluronans.

In some embodiments, the hyaluronans comprise a thiol-modifiedhyaluronan, whose gelation by disulfide bridge formation is triggered inthe presence of polyethylene glycol diacrylate (PEGDA).

In some embodiments, the rheological traits are determined, at least inpart, by a starting concentration and rigidity of thiol-modifiedhyaluronan and PEGDA prior to gelation, a final rigidity of hydrogelpost-gelation achieved by the precise ratios of the volumes ofthiol-modified hyaluronan and PEGDA.

In some embodiments, the first, inner layer exhibits a firstviscoelasticity of from about 50 Pa to about 150 Pa.

In some embodiments, the optional backing contains a hyaluronan hydrogellayer that exhibits a viscoelasticity from about 600 to about 800 Pa.

In some embodiments, the optional third, outer layer comprises ahyaluronan hydrogel layer with viscoelasticity properties of from about200 to about 300 Pa.

In some embodiments, the backing comprises silk.

In some embodiments, the silk backing comprises a purified fibroinofBombyx™ moth silk knitted into a scaffold, including Seri-Silk™ orContour Seri Silk™.

In some embodiments, the epithelial cells comprise biliary tree stemcells (BTSCs) and the mesenchymal cells comprise early-lineage-stagemesenchymal cells (ELSMCs).

In some embodiments, the ELSMCs comprise angioblasts and their immediatedescendants, precursors to endothelia cells, precursors to stellatecells, or combinations thereof.

In some embodiments, the angioblasts express CD117, CD133, VEGFr, but donot express CD31.

In some embodiments, the precursors to endothelia cells express CD133,VEGFr, CD31 and Van Willebrand Factor.

In some embodiments, the precursors to stellate cells express CD146,ICAM-1, alpha-smooth muscle actin (ASMA) and are negative for vitamin A.

In some embodiments, the mixture of epithelial cells and mesenchymalcells is produced by depleting cell suspensions of mature mesenchymalcells, optionally, by repeated panning procedures to remove cells thatattach within from about 15 minutes to about 30 minutes on tissueculture dishes or surfaces at 37° C.

In some embodiments, a culturing of the remaining cell suspensions isperformed on low-attachment dishes and in a serum-free medium until aplurality of organoids is formed by self-assembly of epithelial cellsand mesenchymal cells.

In some embodiments, the serum-free medium comprises a basal medium(with no copper, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serumalbumin (purified, fatty-acid-free; fraction V), 4.5 mM nicotinamide,0.1 nM zinc sulfate heptahydrate, 5 Cpg/ml transferrin/Fe, 5 μg/mlinsulin, and a mixture of purified free fatty acids that are presentedcomplexed with fatty acid free highly purified albumin.

In some embodiments, in which the serum-free medium further comprises 10pig/ml high density lipoprotein.

In some embodiments, in which the plurality of organoids is formed afterabout 2 hours, after about 4 hours, after about 6 hours, after about 8hours, after about 10 hours, after about 12 hours, after about 14 hours,after about 16 hours, after about 18 hours, after about 20 hours, afterabout 22 hours, or after about 24 hours.

In some embodiments, the plurality of organoids comprises BTSCs positivefor:

-   -   a) at least one marker selected from the group of pluripotency        genes consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2 and        Bmi1,    -   b) at least one marker selected from the group of endodermal        transcription factors consisting of Sox9, Sox17, Pdx1,        HNF4alpha, HNFB1 and ONECUT2,    -   c) at least one marker selected from the group of surface        markers associated with stem/progenitors consisting of EpCAM,        NCAM, LGR5, one or more isoforms of CD44, CXCR4, sodium iodide        symporter (NIS), CD49 (integrin A6), CD29 (integrin B1) and        integrin B4;    -   wherein the BTSCs are negative for markers of mature hepatic or        pancreatic cells, including P450s, aquaporin, enzymes involved        in bile production, amylase and digestive enzymes.

In some embodiments, the one or more biomaterial layers compriserecombinant MMPs.

In some embodiments, the one or more biomaterial layers comprise cellsengineered to express MMPs.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains drawings executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofnecessary fee.

FIGS. 1A-1D depicts A) Schematic of the process and estimates of thetime required for preparing organoids, assembling patch grafts and doingthe surgeries. B) Donor cells for the stem cell patch grafts wereisolated from cell suspensions of biliary tree tissue from transgenicpigs with green fluorescent protein (GFP) linked to the H-2B(ACTB-IRES-pH2B-eGFP). The cells were prepared as organoids (floatingaggregates of epithelial and their mesenchymal cell partners) inserum-free Kubota's Medium and on low attachment culture dishes.Organoids of biliary tree stem cells (BTSCs) and of their early lineagestage mesenchymal cell (ELSMCs) partners (angioblasts and precursors toendothelia and to stellate cells) are shown in a phase micrograph versusone demonstrating expression of the transgene, GFP69. Histology of thestem cell organoids that were paraffin embedded, sectioned and stainedwith hematoxylin/eosin. (d) Magnified image of an organoid of BTSC andELSMCs. C) Immunofluorescence (IF) demonstrating expression of stemcell, hepatic and pancreatic markers indicating that these cells areprecursors to both liver and to pancreas as shown in multiple priorarticles and summarized in reviews. D) Representative qRT-PCR assaysassessing expression of various genes in the organoids and indicatingthat cells are stem cells or early progenitors. The controls were maturehepatocytes from piglet livers.

FIGS. 2A-2E depicts A) A schematic of a patch graft affixed to the liverof a pig, and below, the composition of the grafts. Early lineage stagecells, both the epithelia and mesenchymal cells, are required forproduction of matrix metallo-proteinases (MMPs), key regulators of cellmotility, migration and engraftment. Structure of the graft consist oflayers of biomaterials and cells tethered to the target site. For thecells to engraft, the matrix components of the graft biomaterialslocated adjacent to the target site must be soft (˜100 Pa), such ashyaluronan hydrogels. The medium components must be devoid of serum,growth factors and cytokines influential to differentiation of the donorcells and should be ones tailored for survival and expansion of earlylineage stage cells such as stem/progenitors. The backing must havesufficient tensile strength to be used in surgical procedures but beneutral in its effects on the differentiation of the donor cells (oneswith type I collagen should be avoided). The backing is impregnated orcoated with a more rigid 10× hydrogel (˜700 Pa) to serve as a barrier toorient the migration of donor cells towards the target tissue and tominimize adhesions. After attachment to the target site, a 2×HAhydrogel, one that is sufficiently fluid to be coated or painted ontothe outside surface, is added and used to further minimize adhesions. Xindicated the stiffness of the crosslinked hydrogel. B) Graft affixed tothe livers of the hosts. C) Schematic of the graft demonstrating thelayers constituting the graft composition. D) Assays empiricallyassessing the rheological properties (shear and compressive mechanicalforces) of the specific hydrogel layers. E) Analysis of the rheologicalproperties of the 3 layers of hyaluronan hydrogels.

FIGS. 3A-3C depicts A) Masson's Trichrome Staining of the patch graft atone week. Masson's Trichrome identifies collagens (blue), cytoplasm(red) and nuclei (dark purple). It was used to identify Glisson'scapsule (normally adjacent to the liver lobules) and adhesions onoutside surface of the grafts. In the low magnification image (1),Trichrome staining highlighted the frequently observed separation of thehost tissue from the backing and graft in the first weekpost-transplantation due, it is assumed, to the secretion of MMPs. Theliver lobules next to the graft show blanching found subsequently to becaused by penetration of hyaluronans into the tissue. In a highermagnification image (2) of a subsequent section to that in (1), andstained with Hematoxylin/Eosin, there is evidence of a broad region ofremodeling region in which the Glisson Capsule is altered and seeminglyabsent. Higher magnification images of two areas within the remodelingzone make evident the inflammatory responses (a) and the remodeling ofthe liver lobules (b). B) Trichrome staining of the patch graft at threeweeks. The hyaluronans were resorbed enabling maturation of donor cellsaccompanied by muting of expression of MMPs. Inflammation was markedlydecreased leaving a band of tissue between the graft and liverparenchyma (2) and restoration of the liver histology. C) Graft ofporcine BTSCs/ELSMCs tethered to the liver of a NRG/FAH mouse that wasmaintained for 30 days without Nitisinone. Hematoxylin/eo sin staining.

FIGS. 4A-4E depicts A) low magnification image of the patch graft on thesurface of a pig liver after one week. The region in black constitutesthat of the hyaluronans of the graft. Donor GFP±cells (with pink nuclei;white arrows note areas with large numbers of the donor GFP-BTSC/ELSMCs)were visualized by labeling with an antibody to GFP and secondarily withone coupled to Alexa594, a red fluoroprobe. Host nuclei were stainedblue with 4,6-Diamidino-2-phenylindole (DAPI). Host tissue (a) extendsinto the hyaluronans (HA, the black background) of the graft; tissue bythe backing contains occasional organoids but with most donor cellsdispersed into cells that are readily identified as one with pinknuclei. There is no evidence for the usual structure of the Glisson'scapsule in this area that constitutes the region of remodeling. B)Engraftment and migration of donor cells was rapid. Within a week, allidentified donor cells were within the host liver, both near the graftsite and also on the opposite side of the liver lobe (estimated distancefrom the graft site is ˜1.5 cm, a significant distance for the donorcells to have migrated in a week). Shown are donor cells (pink nuclei)near lobules of host mature hepatocytes (forest green color fromauto-fluorescence of lipofuscins) on the distant side of the liver lobefrom that of the graft site, a distance that is approximately 1.5 cm. C)Maturation of donor cells to adult fates occurred in parallel with HAsbeing resorbed. Enlargement of a region containing donor GFP+ cells(single cells with pink nuclei) near to host hepatocytes (a), forestgreen in color (autofluorescence of lipofuscins), and readilydistinguished from mature donor (b) hepatocytes (pink nuclei) andlineage restricted from donor GFP+ stem cells. Some cytoplasmic stainingof the GFP label was observed especially in the first week aftertransplantation but became increasingly nuclear staining by the secondweek (See FIG. 4D-4E). With other IHC assays (data not shown), thespring green color of cells amidst the plates of both host and donorhepatocytes proved by IHC assays to be a mix of mesenchymal cells(endothelia and stellate cells). The bright, spring greenautofluorescence from these cells was readily distinguishable from thatof the dark, forest green of the lipofuscins. D) Examination of porcineliver at 1 week after patch grafting. We used Sirius red stain, an azodye staining collagens; did immunohistochemistry for pan-cytokeratin(pCK) and Sox9; and immunofluorescence (IF) stains performed on serial3-μm sections. At the patch graft site, grafted donor cells merged withliver lobules. In the upper panels (original magnification=5×), patchgrafts are composed of mesenchymal and epithelial pCK+ cells (arrows).In middle panels, a higher magnification is provided (20×). Epithelialcells show an immuno-phenotype that is typical of biliary tree stemcells (BTSCs) expressing biliary cytokeratins (pCK) and the endodermalstem cell marker Sox9. The GFP label in these cells was clearly nuclear.BTSCs within the patch graft are arranged in cell strings reassemblingbile ductules (arrows) and are in direct continuity with hepatocyteplates of the adjacent liver lobule (arrowheads). Hepatocytes in lobulesare pCK and Sox9 negative. In lower panels (Original magnification=20×),the immunofluorescence for GFP allows one to identify individual graftedcells and their progeny. Hepatocytes in lobules adjacent to the patchgraft were GFP positive, some in nucleus and some cytoplasmically,indicating that these were donor cells that had integrated amidst hostliver parenchyma. At the interface between patch graft and liverlobules, pCK+/GFP+ ductules were in direct continuity with GFP+/pCK−cells within the lobules (arrowheads) suggesting a maturation ofgrafting cells towards an hepatocyte fate. E) Examination of porcinelivers 2 weeks after patch grafting. IF stains reveal that GFP+ cellsare present within lobules distant to the graft site and co-expressmature hepatocyte markers such as Hepatocyte Nuclear Factor (HNF) 4a andalbumin. The GFP label was primarily nuclear in the donor cells.Separate or merged channels were included. Nuclei were displayed in blue(DAPI). Original Magnification: 40×

FIGS. 5A-5P depicts rescue of NRG/FAH mice from type I tyrosinemia witha patch graft of porcine BTSCs/ELSMCs. The proof of entry of the donorcells into the liver is provided in FIG. 3C. Patch grafts of porcineBTSCs/ELSMCs were applied surgically to the liver of NRG/FAH mice andthen provided with regular water (weaning them from Nitisinone). FIG. 11shows a survival chart of the mice along with the body weights of theanimals. Whereas the experimentals, provided patch grafts, were aliveand increasing in body weight after 30 days, the controls begandeclining after 2 weeks and had to be euthanized by day 17. The kidneysof the animals provided patch grafts with cells were normal (FIG. 5N).FIGS. 5A-5B) depict positive control NRG/FAH mouse liver in host treatedwith Nitisone (NTBC) as visualized by low and high magnification imageof H&E stained section of the liver. FIGS. 5C-5D) depict negativecontrol mice with patch graft with no cells as visualized by low andhigh magnification image of H&E stained section of NRG/FAH mouse liverweaned from NTBC treatment and evaluated at day 16. FIGS. 5E-5F) depictlow and high magnification images of section of liver of NRG/FAH mouseliver patch grafted with porcine BTSCs/ELSMCs (see also FIG. 3C). FIGS.5G-5H) depict low and high magnification images of grafted liver stainedfor GFP (using antibody to GFP and coupled to Novo Red). Note that theGFP+ cells are present throughout the liver and the GFP is localizedprimarily to the nucleus (FIG. 5H). FIG. 5I depict images showinglocalization of GFP linked to Histone H2B. Note that expression of GFP(linked to Histone H2B) is present both in the nucleus and in thecytoplasm of some cells. FIG. 5P depicts immunofluorescence imagesshowing faint cytoplasmic labelling with antibody to histone H2B. FIGS.5J-5K) depicts low and high magnification image of control sectionsprepared without the primary antibody. FIGS. 5L-5M) depict controls forthe studies on murine liver (NRH/FAH) patch grafted with porcineBTSCs/ELSMCs. FIG. 5L depicts a negative control for goat antibodies;FIG. 5M depicts a aegative control for rabbit antibodies. FIG. 5Ndepicts images of Kidney from a NRH/FAH mouse with patch graft onto theliver with (1) being the graft with no cells and (2) being one withBTSCs/ELSMCs. FIG. 5O depicts murine liver (NRH/FAH) patch grafted withporcine BTSCs/ELSMCs for porcine furamyl-aceto-acetate hydrolase (FAH),and for histone H2B as shown in FIG. 5P. Note that the histone, H2B, isfound predominantly in the nucleus but is also found cytoplasmically insome cells.

FIGS. 6A-6H depicts isoforms of secreted and membrane-associated MMPsexpressed by both epithelial stem/progenitors and their partner ELSMCsversus that in various mature cells in liver. Quantitation of theexpression levels indicated that the membrane-associated forms weresimilar for both stem/progenitors/ELMCs and for matureepithelial/mesenchymal partners. Note the comparisons in (D). Bycontrast, secreted forms were expressed at very high levels instem/progenitors/ELMCs and at low or negligible levels in mature celltypes. The cell populations of adult cells analyzed were isolated fromsuspensions of piglet livers and biliary tree tissue and comprised ofCD45+ cells (hemopoietic cells), CD146+ cells (stellate cells), CD31+cells (endothelia), EpCAM+/CD45− cells (committed progenitors anddiploid hepatocytes and cholangiocytes). The BTSCs were isolated fromthe biliary tree by the protocols given in the Methods. FIG. 6A) showsMMP expression in BTSCs. FIG. 6B) shows MMP expression in stellate cellsand endothelia. FIG. 6C) shows MMP expression in 2N (diploid)hepatocytes. FIG. 6D) shows representative MMPs in Adult Cell Typesversus BTSCs. FIG. 6E) shows representative MMP expression in regions ofremodeling with a BTSC/ELSMCs graft. In a section adjacent to the patchgraft of BTSCs/ELSMCs was stained with Trichrome indicating the region(bracketed) of remodeling. FIG. 6F) shows representative images of IHCassays for MMP1 (Novo-red+). Methyl green as the background stain. FIG.6G) Section stained for MMP2 (Novo-red+). Hematoxylin as backgroundstain. Within the liver lobules, the remodeling process included platesor fingers of MMP2+ cells (rust colored) that transitioned to a completeloss of lobular structures. With clearance of HAs, the lobularstructures reappeared.

FIG. 7 depicts a schematic demonstration of the engraftment andintegration phenomena in the liver and of the pancreas. Patch graftswere tethered to the liver via sutures at the corners of the patch. Tominimize adverse reactions (e.g. autolysis) by the pancreas, the patchgrafts tethered to the pancreas were done entirely with surgical glue atthe corners of the patch (mice) or were done with sutures to theduodenum on one end of the patch and using surgical glue on the cornerson the other end, that over the pancreas (pigs)

FIGS. 8A-8B depicts controls of transgenic pig liver versus wild typepig liver. Both are unstained frozen sections and imaged for greenfluorescence. High magnification images of a frozen section of liverfrom a transgenic pig (8A), with GFP+-H2B fluorescence that is punctatedue to nuclear localization. However, note the variations in the levelsof fluorescence among different cells. By contrast, a frozen sectionfrom the liver of a wild type pig (8B) demonstrates green fluorescencederived from autofluorescence of lipofuscins and other components and isclearly cytoplasmic.

FIGS. 9A-9B depicts the remodeling zone near to graft site. In the firsttwo weeks after transplantation, the region adjacent to the graftundergoes extensive remodeling of the Glisson Capsule and of thesubjacent lobular architecture of the liver parenchyma. See also FIG. 3.Images made at one week (9A) and two weeks (9B) after grafting and withhigher magnification images of specific regions of the remodeling zone.Stain: hematoxylin/eosin.

FIG. 10 depicts adverse conditions with patch grafts with certainbackings. The adverse reactions included necrosis, adhesions, and sitesof cholestasis found to occur when grafts were placed too close to someducts such that the swelling caused occlusion of the ducts.

FIG. 11 depicts viability and weight chart of NRG/FAH mice patch graftedwith porcine BTSCs/ELSMCs and then weaned from the drug, Nitisinone(NTBC). The chart begins with animals after weaning from the drug. Theanimals (n3) with patch grafts were stable and, indeed gained weightover the 30 days of the study. By contrast, the controls (n2) werestable temporarily but by about two weeks began to decline. They had tobe euthanized by day 16.

FIGS. 12A-12E depicts adverse conditions with patch grafts with certainbackings. The adverse reactions included necrosis (12A), discoloration(12B), and adhesions (12C). Difficulties that proved independent of thegraft biomaterials and composition were skin infections (12D, 12E). Theskin infections proved a common problem due to the immunosuppression.The infections were not observed within the peritoneum or associatedwith the grafts, but only in association with the skin.

FIGS. 13A-13C shows a frozen section from the liver of a NRG/FAH mousegiven a patch graft of porcine GFP+ BTSCs/ELSMCs (FIG. 13A). The micewere evaluated one month after transplantation. The section was stainedwith DAPI but not with a primary antibody. The generalized pale greenbackground is from autofluorescence of various components in liver. Bar:500 μm. Mice subjected to patch grafts of porcine BTSCs/ELSMCs andevaluated one month later. FIG. 13B depicts a frozen section showing theoccasional host cells (blue nuclei and yellow/green cytoplasm) in tissuedominated by donor cells (red/purplish nuclei). Bar: 500 μm. FIG. 13Cdepicts a higher magnification image of 13B and with labeling toindicate the occasional mouse cell in tissue dominated by donor(porcine, GFP+) cells. Bar: 100 μm

FIG. 14 depicts the rapid distribution and integration of the donorcells throughout the host tissue. The schematic figure indicates theregions of the liver of a piglet and that were sampled a week after apatch graft of GFP+ BTSCs was surgically applied to the piglet liver.The different regions of the piglet liver were used to prepare DNA PCRAssays for GFP expression in each region. The agarose gel shows theresults of the DNA PCR assay for GFP expression in each of these zones

FIGS. 15A-15D depicts data are from serological studies in mice providedpatch grafts with murine organoids of BTSCs/ELSMCs from DS-red mice.Controls received grafts without cells. The patch grafts were attachedto the pancreas of NRG/Akita mice, a murine model of type I diabetes.The number of mice for the control groups (untreated or treated withbiomaterial without cells) were 3; those for the diabetic mice treatedwith patch graft were 4 and 5. A) depicts the blood glucose levels inuntreated diabetic mice (circle), diabetic mice treated with graftbiomaterial only (square), and diabetic mice treated with a patch graftcontaining cells (triangle). B) depicts the serum levels of C-peptide inuntreated diabetic mice (see first column under D0, D7, D14, and D21),diabetic mice treated with graft biomaterial only (see second columnunder D0, D7, D14, and D21), and diabetic mice treated with a patchgraft containing cells (see third column under D0, D7, D14, and D21). C)depicts serum insulin levels in untreated diabetic mice (see firstcolumn under D21 and D28), diabetic mice treated with graft biomaterialonly (see second column under D21 and D28), and diabetic mice treatedwith a patch graft containing cells (see third column under D21 andD28). D) depicts blood glucose levels in normal mice (triangles),diabetic mice treated with graft biomaterial only (circles), anddiabetic mice treated with a patch graft containing cells (squares).

FIGS. 16A-16B shows representative light micrographs of sections ofmurine pancreas stained with antibodies to insulin (brown) andcounterstained with hematoxylin (same magnification, bar=100 μm). (A)Diabetic mice treated with patch graft containing organoids ofBTSC/ELSMCs prepared from DS-red mice. (B) Diabetic mice treated with agraft without cells. Note that there are many cells not expressinginsulin. Even though the insulin protein is evident in the controls, itwas not functional as noted in the serological assays (FIG. 15). Theimmunohistochemistry was prepared on tissues one month aftertransplantation with patch grafts.

FIGS. 17A-17C depicts Akita/NRG mice given patch grafts of organoids ofBTSCs/ELSMCs prepared from DS-red mice. A) depicts representativephotomicrographs show islets or patch area with immunoperoxidase(brown)-stained Neurogenin-3 (NGN3), counterstained with hematoxylin,initial magnification 40×, bar=100 μm), diabetic mice treated withorganoids in patch graft. B) depicts antibody-positive organoids in andaround the patch region (arrows). C) depicts an islet in Akita/NRGdiabetic mice treated with a patch graft without cells. Note that thereis minimal or no expression of NGN3.

FIGS. 18A-18B depicts representative photomicrographs show islets orpatch area with immunoperoxidase (brown)-stained Neurogenin-3,counterstained with hematoxylin, initial magnification 40×, bar=100 μm.(A) depicts diabetic mice given patch graft with organoids ofBTSCs/ELSMCs prepared from DS-red mice. A few DS-red-positive stainingcells (indicative of donor cells) can be observed in the pancreaticparenchyma (black arrows). (B) some antibody-positive organoids can beseen as indicated by the narrow (brown arrows). Note: The wider (blue)arrows point to the silk fibers in the graft.

FIGS. 19A-19B shows evidence for Engraftment of organoids of BTSCs/ELMCsfrom GFP+ transgenic pigs into the pancreas of wild type pigs by a weekafter the surgery. The image shows low magnification images of a pigpancreas section stained with DAPI (4′,6-Diamidine-2′-phenylindoledihydrochloride) and for immunofluorescent staining for insulin (A)versus amylase (B) in a section of piglet pancreas and surroundingtissues a week after attaching a patch graft containing organoids ofporcine GFP+ BTSCs/ELSMCs onto the pancreas of a wild type piglet. Thestaining for insulin was done with an antibody coupled to a redfluoroprobe and that for GFP with one with a green fluoroprobe. Notethat cells have migrated also into the submucosa of the duodenum, thelocation of Brunner's Glands. A) depicts large number of GFP+donor-derived cells are visible in the proximity of the area where thepatch graft was placed. Nuclei were stained with DAPI and appear blue.Insulin expression, characteristic of pancreatic islet beta cells, wasidentified with an anti-insulin antibody coupled to a red fluoroprobe.Endogenous (host) islet beta cells of the recipient pancreas appear red,in the upper portion of the pancreas. Donor-derived islet beta cellshave nuclei that are red/purple from the merge of DAPI's blue and theGFP's green and have cytoplasm that is red/yellow from staining forinsulin. They are observed in the lower portion of the pancreas,proximal to the site of placement of the patch graft. This lowmagnification image demonstrates the extent of engraftment into thepancreas, as well as the engraftment into the submucosal region of theduodenum, the location of Brunner's Glands (hypothesized to be thestarting point of the network of cells contributing to organogenesis ofthe liver and pancreas). Bar: 1 mm. B) shows immunofluorescent stainingfor amylase in a sequential section from the same tissue block as thatin FIG. 18A. Amylase (green) is detected predominantly in pancreaticacinar tissue, as well as in the mucosal layer and in the lumen of theduodenum. Insulin (red) does not overlap with Amylase (green). Thisstaining, in combination with that of FIG. 19A, suggests that a largeportion of GFP+ donor-derived cells are committed to a pancreaticacinar-like fate. Bar: 1 mm.

FIGS. 20A-20C depicts sections stained with DAPI (blue) andimmunofluorescent staining for insulin and other islet hormones and forGFP in pancreas sections from 3 recipients of GFP+ BTSCs/ELSMCs patchgrafts (day 7 post transplantation). We observed GFP+ cells in theparenchyma of the pancreas, in the proximity of the patch graft site inall recipients. Notably, GFP+ cells appear at a considerable distancefrom the patch graft site and appear to be well integrated into theparenchyma of the recipient pancreas. Patch graft material (SERI silk)presents a degree of autofluorescence in different channels and is stillvisible at day 7 post transplantation. Bar: 2 mm. FIG. 20A is a highermagnification of FIG. 19A.

FIGS. 21A-21C show higher magnification images, corresponding to thesections in FIGS. 20A-20C. FIGS. 21A-21C depicts evidence showing theco-existence of endogenous host islet-beta cells (Insulin+/GFP−: redcytoplasm) and donor-derived beta cells (Insulin+/GFP+: purplish colorednuclei and red/orange cytoplasm) in the pancreas at 7 dayspost-transplantation of GFP+ BTSCs/ELSMCs patch graft. We observeddonor-derived as well as endogenous host islet-beta cells in all cases.The majority of GFP+ cells present a phenotype consistent with that ofpancreatic acinar cells. GFP+ cells organized to form a ductal structureare visible in the lower portion of FIG. 21A. Although the islet cellsshow GFP expression nuclearly even at a week, GFP expression in theacinar cells at a week was cytoplasmic. As was observed in FIGS. 4A-4Ein the first week after attaching patch grafts onto liver, there iscytoplasmic staining for GFP; in these images, there is GFP stainingcytoplasmically in the acinar cells (blue nuclei and green cytoplasm).In the liver, this resolved to entirely nuclear staining for GFP byabout 2 weeks post-transplantation. Ongoing studies are assessing ifthis occurs also in the acinar cells in the pancreas. Bar: 50 μm.

FIG. 22 shows a graphical outline of different stem cell subpopulations.

DETAILED DESCRIPTION

Described herein are novel patch graft compositions and methods fortransplantation of cells into tissue and solid organs.

Embodiments according to the present disclosure will be described morefully hereinafter. Aspects of the disclosure may, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Theterminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. All publications, patent applications,patents and other references mentioned herein are incorporated byreference in their entirety.

Definitions

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. While not explicitlydefined below, such terms should be interpreted according to theircommon meaning.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology, and recombinant DNA,which are within the skill of the art. See, e.g., Sambrook and Russelleds. (2012) Molecular Cloning: A Laboratory Manual, 4rd edition; theseries Ausubel et al. eds. (2012) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (2014) Antibodies, A Laboratory Manual,2d edition; Freshney (2011) Culture of Animal Cells: A Manual of BasicTechnique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S.Pat. No. 4,683,195; Hames and Higgins eds. (1985) Nucleic AcidHybridization; Anderson (1999) Nucleic Acid Hybridization; Hames andHiggins eds. (1984) Transcription and Translation; Immobilized Cells andEnzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to MolecularCloning; Miller and Calos eds. (1987) Gene Transfer Vectors forMammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003)Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds.(1987) Immunochemical Methods in Cell and Molecular Biology (AcademicPress, London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5%, or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about.” It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount.

The terms or “acceptable,” “effective,” or “sufficient” when used todescribe the selection of any components, ranges, dose forms, etc.disclosed herein intend that said component, range, dose form, etc. issuitable for the disclosed purpose.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. As used herein, the transitional phrase “consistingessentially of” (and grammatical variants) is to be interpreted asencompassing the recited materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the recitedembodiment. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” as used herein should not beinterpreted as equivalent to “comprising.” “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions disclosed herein.Aspects defined by each of these transition terms are within the scopeof the present disclosure.

As used herein the term “amount effective” or “effective amount” refersto an amount that is sufficient to treat disease states or conditions(e.g. liver or pancreatic diseases). An effective amount can beadministered in one or more administrations, applications or dosages.Such delivery is dependent on a number of variables including the timeperiod during which the individual dosage unit is to be used, thebioavailability of the composition, the route of administration, etc. Itis understood, however, that specific amounts of the compositions forany particular patient depends upon a variety of factors including theactivity of the specific agent employed, the age, body weight, generalhealth, sex, and diet of the patient, the time of administration, therate of excretion, the composition combination, severity of theparticular disease (e.g. liver or pancreatic disease) being treated andform of administration.

The terms “equivalent” or “biological equivalent” are usedinterchangeably when referring to a particular molecule, biological, orcellular material and intend those having minimal homology while stillmaintaining desired structure or functionality.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample; further, the expressionlevel of multiple genes can be determined to establish an expressionprofile for a particular sample.

As used herein, the term “functional” may be used to modify anymolecule, biological, or cellular material to intend that itaccomplishes a particular, specified effect.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three dimensional (3D) structureand may perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers.

A polynucleotide can comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure can be imparted before or after assembly of thepolynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double and single stranded molecules.Unless otherwise specified or required, any aspect of this technologythat is a polynucleotide encompasses both the double stranded form andeach of two complementary single stranded forms known or predicted tomake up the double stranded form.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another aspect, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

The term “gene” as used herein is meant to broadly include any nucleicacid sequence transcribed into an RNA molecule, whether the RNA iscoding (e.g., mRNA) or non-coding (e.g., ncRNA). The term “isolated” asused herein refers to molecules or biologicals or cellular materialsbeing substantially free from other materials.

Definitions of other terms used herein will be provided below in thecontext in which they are used.

Patch Graft Compositions and Strategies

In another aspect, the present disclosure relates to a patch graftcomprising a mixture of epithelial cells and mesenchymal cells and oneor more biomaterial layers including, at least:

-   -   a first, inner layer for contacting a solid organ, the first        inner layer exhibiting a first viscoelastic property,        incorporating a mixture of epithelial cells and mesenchymal        cells, supporting an ability of the epithelial cells and the        mesenchymal cells to produce secreted matrix metallo-proteinases        (MMPs) and promoting viability and immaturity of said epithelial        cells and mesenchymal cells;    -   optionally, a backing that confers a barrier to the cells        migrating in a direction other than towards the solid organ, the        backing exhibiting a second viscoelastic property; and    -   optionally, a third, outer layer of a coating or material that        minimizes adhesions of the patch graft to internal surfaces of a        body cavity, including internal walls and/or organs, in        proximity to the patch graft; wherein said viscoelastic        properties are determined by measuring rheological traits and        expressed in Pascals (Pa).

As used herein, the term “patch graft” refers to a composition of cellsembedded in an appropriate biomaterial that allows for transplantingdonor cells to the host. In some embodiments, the term refers to acomposition of cells embedded in an appropriate biomaterial that allowsfor transplanting donor cells to the host. Biomaterials are ones thatcan be prepared under wholly defined conditions (e.g., basal medium ofnutritional factors, vitamins, amino acids, carbohydrates, minerals,insulin, transferrin/Fe, and/or lipids) and solidified into a soft gel(about 100 Pa), and covered with a backing that has sufficient tensilestrength to enable surgical attachment to a tissue or organ of the hostand yet be of a chemistry with minimal effects on the differentiation ofthe donor cells and minimal adverse effects to the host tissues.

As used herein, the term “backing” refers to a material which (i) isbiocompatible with the subject into which it is being transplanted, (ii)exhibits mechanical resilience to withstand the compressive and shearforces that occur on organs and tissues (especially internal ones),which in turn enables this material to function as a surgical tissue,and (iii) has a neutral or minimal effect on the differentiation statusof cells that come in contact with the material. In this regard,suitable materials include but are not limited to Seri-Silk™ (orderivatives of it) and/or a patch comprised of PGA and/or PLLA.Non-limiting examples of suitable patches of synthetic materials includea woven patch comprised of 91% PGA-co-9% PLLA, a knit patch comprised of91% PGA-co-9% PLLA, or a non-woven patch comprised of 100% PGA. Otherpossibilities include amnions or matrix extracts of amnions or omentumor matrix extracts of omentum. In some embodiments, the backingcomprises silk. In some embodiments, the backing comprises silk. In someembodiments, the silk backing comprises a purified fibroin of Bombyx™moth silk knitted into a scaffold, including Seri-Silk™ or Contour SeriSilk™.

In some embodiments, the backing is also bioresorbable. As used herein,“bioresorbable” refers to a material that can be broken down by the bodyof a host or recipient of the graft and does not require mechanicalremoval. In some embodiments, the bioresorbable backing is bioresorbablewithin a span of about 2 to about 10 weeks, about 2 to about 20 weeks,about 2 to about 52 weeks, about 4 to about 16 weeks, about 4 to about12 weeks, or about 4 to about 8 weeks.

As used herein, the biomaterials of the graft, and independent of thebacking, include ones that can form hydrogels. The term “gel” refers toa solid jelly-like material that can have properties ranging from softand weak to hard and tough. Gels are defined as a substantially dilutecross-linked system, which exhibits no flow when in the steady-state. Byweight, gels are mostly liquid, yet they behave like solids due to athree-dimensional cross-linked network within the liquid. It is thecrosslinking within the fluid that gives a gel its structure (hardness)and contributes to its adhesiveness. In this way gels are a dispersionof molecules of a liquid within a solid in which the solid is thecontinuous phase and the liquid is the discontinuous phase. A “hydrogel”is a non-limiting example of a gel comprised of a macromolecular polymergel constructed of a network of polymer chains. Hydrogels aresynthesized from hydrophilic monomers or hydrophilic dimers (e.g. in thecase of hyaluronan) by either chain or step growth, along with networkformation. A net-like structure along with void imperfections enhancethe hydrogel's ability to absorb large amounts of water via hydrogenbonding. As a result, hydrogels develop characteristic firm yet elasticmechanical properties. They are able to undergo spontaneous formation ofnew bonds when old bonds are broken within a material. The structure ofthe hydrogels along with electrostatic attraction forces drive new bondformation through non-covalent hydrogen bonding. One successful materialfor the hydrogels is thiol-modified hyaluronan that can be triggered toform hydrogels when exposed to oxygen and/or to poly (ethylene glycol)diacrylate (PEGDA) and readily “tunable” by the precise ratios ofhyaluronan and PEGDA concentrations (and/or oxygen levels). Thehydrogel's viscoelastic properties can be determined by measuringrheological traits and expressed in Pascals (Pa).

In some embodiments, the second viscoelastic property (expressed in Pa)has a greater value than the first viscoelastic property. In someembodiments, the one or more biomaterial layers comprise a hydrogel,which further comprises minimally sulfated or non-sulfatedglycosaminoglycans. In some embodiments, the non-sulfatedglycosaminoglycans comprise hyaluronans. In some embodiments, thehyaluronans comprise a thiol-modified hyaluronan, whose gelation bydisulfide bridge formation is triggered in the presence of polyethyleneglycol diacrylate (PEGDA). In some embodiments, the rheological traitsare determined, at least in part, by a starting concentration andrigidity of thiol-modified hyaluronan and PEGDA prior to gelation, afinal rigidity of hydrogel post-gelation achieved by the precise ratiosof the volumes of thiol-modified hyaluronan and PEGDA.

In some embodiments. the rheological trails of the patch graft are alsodetermined by the temperature for achieving the cross-linking process.Temperatures for forming about 50 Pa to about 150 Pa inner layerhydrogel can be room temperature (RT) or 37° C. Temperatures for formingabout 200 to about 300 Pa hydrogel can be 4° C. or room temperature(RT). Temperatures for forming about 600 to about 800 Pa can be roomtemperature (RT) or 37° C.

As used herein, the term “hyaluronan,” or “hyaluronic acid,” refers to apolymer of disaccharide units comprised of glucosamine and glucuronicacid [1-3] linked by (31-4, (31-3 bonds and salts thereof. Thus, theterm hyaluronan refers to both natural and synthetic forms ofhyaluronans. The naturally occurring hyaluronan (HA), water-solublepolysaccharide comprising disaccharide units of D-glucuronic acid(GlcUA) and N-acetyl-D-glucosamine (GlcNAc), which are alternatelylinked, forming a linear polymer. High molecular weight HA may comprise100 to 10,000 disaccharide units. HAs often occur naturally as thesodium salt, sodium hyaluronate. HA; sodium hyaluronate, andpreparations of either HA or sodium hyaluronate are often referred to as“hyaluronan.” Non-limiting examples of acceptable hyaluronate salts,include potassium hyaluronate, magnesium hyaluronate, and calciumhyaluronate.

Other glycosaminoglycans (GAGs) can also be used in the hydrogel. Theseinclude forms of chondroitin sulfate (CSs) and dermatan sulfates (DSs),polymers of glucuronic acid and galactosamine, and heparan sulfates(HSs) and heparins (HPs), polymers of glucuronic acid and glucosamine.The extent and pattern of sulfation of these GAGs is critical, since thesulfation patterns dictate the formation of complexes with multiplefamilies of proteins (e.g. coagulation proteins, growth factors,cytokines, neutrophilic enzymes). See, e.g., Powell A K, Yates E A,Fernig D G, Turnbull J E. Interactions of heparin/heparan sulfate withproteins: appraisal of structural factors and experimental approaches.Glycobiology. 2004 April; 14(4):17R-30R. Those appropriate for patchgrafts that optimize engraftment comprise hyaluronans, non-sulfatedGAGs, and ones with minimal sulfation such as forms of chondroitinsulfates found in stem cell niches, as shown in Karumbaiah L, et al.Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Nichesfor Neural Stem Cells. Bioconjug Chem. 2015 Dec. 16; 26(12):2336-49 andHayes A J, et al. Chondroitin sulfate sulfation motifs as putativebiomarkers for isolation of articular cartilage progenitor cells. JHistochem Cytochem. 2008 February; 56(2):125-38 (incorporated herein byreference).

The hydrogel's viscoelastic property may be from about 10 Pa to about 50Pa, from about 50 Pa to about 100 Pa, from about 100 Pa to about 150 Pa,from about 150 Pa to about 200 Pa, from about 200 Pa to about 250 Pa,from about 250 Pa to about 300 Pa, from about 300 Pa to about 350 Pa,from about 350 Pa to about 400 Pa, from about 400 Pa to about 450 Pa,from about 450 Pa to about 500 Pa, from about 500 Pa to about 550 Pa,from about 550 Pa to about 600 Pa, from about 600 Pa to about 650 Pa,from about 650 Pa to about 700 Pa, from about 700 Pa to about 750 Pa,from about 750 Pa to about 800 Pa, from about 800 Pa to about 850. Insome embodiments, the viscoelastic property of the hydrogel is about 50Pa, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa, about 300Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500, Pa, about 550Pa, about 600 Pa, about 650 Pa, about 700 Pa, about 750 Pa, or about 800Pa.

In some embodiments, the first, inner layer exhibits a firstviscoelasticity of from about 50 Pa to about 150 Pa. In someembodiments, the optional backing contains a hyaluronan hydrogel layerthat exhibits a viscoelasticity from about 600 to about 800 Pa. In someembodiments, the optional third, outer layer comprises a hyaluronanhydrogel layer with viscoelasticity properties of from about 200 toabout 300 Pa. In some embodiments of the patch graft, theviscoelasticity properties of the backing is greater than theviscoelasticity properties of the first layer. In some embodiments ofthe patch graft, the viscosity of the backing is about 1.5 to about 15fold greater than the viscosity of the first layer. In some embodimentsof the patch graft, the viscoelasticity properties of the backing isabout 2 fold greater than the viscoelasticity properties of the firstlayer.

Applicants have shown that hyaluronans can influence epithelial cells,stem and/or progenitor cells to express factors that regulate criticalcell adhesion molecules needed for cell attachment and cell-cellinteractions and to prevent the stem and/or progenitor cells frominternalization of those attachment factors following cell suspensionpreparations, cryopreservation, or with transplantation. Non-limitingexamples of such attachment factors include integrins. Integrins are alarge family of heterodimeric transmembrane glycoproteins that functionto attach cells to extracellular matrix proteins of the basementmembrane, ligands on other cells, and soluble ligands. Integrins containa large and small subunit, referred to as α and β, respectively. Thissubunits form αβ heterodimers and at least 18 α and eight β subunits areknown in humans, generating 24 heterodimers. In some embodiments, thestem and/or progenitor cells express higher levels of integrin subunits,for example, ITGα1, ITGα2, ITGα2B, ITGα3, ITGα4, ITGα5, ITGα6, ITGα7,ITGα8, ITGα9, ITGα10, ITGα11, ITGαD, ITGαE, ITGαL, ITGαM, ITGαV, ITGαX,ITGβ1, ITGβ2, ITGβ3, ITGβ4, ITGβ5, ITGβ6, ITGβ7 and ITGβ8. In onepreferred embodiment, the stem and/or progenitor cells express higherlevels of integrin subunit beta 1 (ITGβ1) and/or integrin subunit beta 4(ITGβ4). Takada Y. et al. (2007) Genome Biol. 8(5): 215.

In some embodiments, the epithelial cells, stem and/or progenitor cellsof the present disclosure differ from naturally occurring stem and/orprogenitor cells in at least that they express an integrin subunit in anamount that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200% greater than theamount of the integrin subunit in unmodified stem and/or progenitorcells. It is contemplated that an increase in an integrin subunit helpthe stem and/or progenitor cell to attach, and to form cell-cellinteractions.

Hyaluronans (HA), major constituents of stem cell niches, are candidatecoatings of stem cells used for cell therapies, since they facilitateviability, proliferation and engraftment in damaged livers. The chemicaland mechanical properties of HA are conducive to essential requirementsfor stem cells. In addition, since the liver is a primary site for HAclearance, HA-coating represents an advantageous strategy for theselective targeting of the transplanted cells to the liver.

The stem and/or progenitor cells can be coated with hyaluronan (HA)using any method known in the field. For example, the stem and/orprogenitor cells can be incubated with an amount of HA and gently mixedfor a period of time, for example, about 5 minutes, 10 minutes, 15minutes, 20 minutes, 30 minutes, 45 minutes, an hour, or longer. TheHA-coated stem and/or progenitor cells can then be further incubated ina medium, for example, Kubota's medium.

In some embodiments, the HA comprises a salt of HA, for example, analkali metal salt. HA often occurs naturally as the sodium salt, sodiumhyaluronate. Non-limiting examples of acceptable additional hyaluronatesalts, include, potassium hyaluronate, magnesium hyaluronate, andcalcium hyaluronate. To prepare the HA for coating the stem and/orprogenitor cells, the HA can be resuspended in any pharmaceuticallyacceptable carrier, for example, phosphate buffered saline (PBS), abasal media, Kubota's Medium, a hormonally-defined medium, or the like.In some embodiments, the pharmaceutically acceptable carrier furthercomprises one or more growth factors, one or more glucosaminoglycansaccharides, or combinations thereof.

In some embodiments, the amount of hyaluronan in the pharmaceuticallyacceptable carrier is from about 0.05% w/v to about 1% w/v hyaluronan.In one preferred embodiment, the amount of hyaluronan in thepharmaceutically acceptable carrier is about 0.1% w/v hyaluronan.

As used herein, the term “coated” means either continuous ordiscontinuous, i.e., the hyaluronan (HA) coating can completely coverthe surface of the stem and/or progenitor cells or covers only a portionso that it forms areas of coverage (e.g. “islands”) and areas of nocoverage. While the coatings of the present invention contain HA, it iscontemplated that the coatings can also comprise other substances. Insome embodiments, the hyaluronan coats at least a portion of a surfaceof said stem and/or progenitor cells or aggregates thereof, for example,at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, atleast 40%, at least 30%, at least 20%, or at least 10% of a surface ofsaid stem and/or progenitor cells or aggregates thereof. In someembodiments, at least about half of an exposed surface of saidaggregates or individual stem and/or progenitor cells is coated withhyaluronan or a pharmaceutically acceptable salt thereof. In someembodiments, the stem and/or progenitor cells have been modified by thepresence of externally added hyaluronan.

The size of transplanted cells may be an important factor for thesuccess of the transplantation. If the cells are too large (e.g.polyploid hepatocytes) or if they form large aggregates, thetransplantation of them via a vascular route may result in an embolusthat can be life threatening. If the cells are too small, theirengraftment efficiency may be very low, and the cells will have agreater propensity to distribute to ectopic sites. Both possibilitiesare ones of importance for cell therapy considerations. Cells used forcell therapy of liver diseases have been infused into the liver via thespleen in animal models or into the portal vein or hepatic artery inhumans. The sizes in terms of the cell diameters have ranged from ˜8-10μm for stem cells (HpSCs, BTSCs), ˜12-15 for hepatoblasts and committedprogenitors, ˜17-18 μm for diploid hepatocytes dominant in neonatallivers, to ˜25-30 μm for mature hepatocytes that are dominant in adultliver.

In some embodiments, a majority of aggregates of stem and/or progenitorcells comprise between about two to about ten stem and/or progenitorcells per aggregate. Most commonly, they comprise 50-100cells/aggregate. In one embodiment, the aggregates comprise about 90-100cells. In one embodiment, the aggregates comprise about 80-90 cells. Inone embodiment, the aggregates comprise about 70-80 cells. In oneembodiment, the aggregates comprise about 60-70 cells. In oneembodiment, the aggregates comprise about 50-60 cells.

In one embodiment, the aggregates comprise about five stem and/orprogenitor cells and associated ELSMCs. In some embodiments theaggregates of stem and/or progenitor have an average diameter of 50 μmor less, 45 μm or less, 40 μm or less, 35 μm or less, 30 μm or less, 25μm or less, 20 μm or less, 15 μm or less, or 10 μm or less. In onepreferred embodiment, the aggregates of stem and/or progenitor cellshave an average diameter of 30 μm or less.

As used herein, the term “cell” refers to one or more cells of thegraft. The cells of the present disclosure are eukaryotic. In someembodiments, the epithelial cells comprise early lineage stage epitheliacells (ELSEs) and the mesenchymal cells comprise early lineage stagemesenchymal cells (ELSMCs), or in which the epithelial and mesenchymalcells are of later lineage stages but are of comparable lineage stagesas each other. In some embodiments, the ELSMCs comprises angioblasts,precursors of endothelia, stellate cells, or combinations thereof. Insome embodiments, the ELSEs and/or the ELSMCs are derived from embryonicstem (ES) cells or from induced pluripotent stem cells (iPS). In someembodiments, the epithelial cells are of a later lineage stage and themesenchymal cells are early lineage stage mesenchymal cells (ELSMCs), orthe mesenchymal cells are of a later lineage stage and the epithelialcells are early lineage stage epithelial cells (ELSEs). In someembodiments, this cell is of animal origin and can be a stem cell, amature somatic cell, progenitor cell, or intermediates in the lineagestages from the stem cells to the mature cells. The term “population ofcells” refers to a group of one or more cells of the same or differentcell type with the same or different origin. In some embodiments, thispopulation of cells may be derived from a cell line, from freshlyisolated cells, or in some embodiments, this population of cells may bederived from a portion of an organ or tissue.

The term “stem cell” refers to cell populations that can self-replicate(produce daughter cells identical to the parent cell) and that aremultipotent, i.e. can give rise to more than one type of adult cell. Theterm “progenitor cell” or “precursor” as used herein, is broadly definedto encompass progeny of stem cells and their descendants. Progenitorsare cell populations that can be multipotent, bipotent, or unipotent buthave minimal (if any) ability to self-replicate. Committed progenitorsare ones that are unipotent and can differentiate into a particularlineage leading to only one mature cell type. Non-limiting examples ofstem cells include but are not limited to embryonic stem (ES) cells,induced pluripotent stem (iPS) cells, germ layer stem cells, determinedstem cells, perinatal stem cells, amniotic fluid-derived stem cells,mesenchymal stem cells (MSCs), and angioblasts. Intermediates betweenstem cells and committed progenitors include cell populations such ashepatoblasts and pancreatic ductal progenitors and other forms oftransit amplifying cells that may be multipotent and have extensiveproliferative potential but more limited (if any) self-replicativeability.

The cells can be any determined stem cells such as determined endodermalstem cells or progenitors derived from them. Moreover these determinedstem cells or progenitors can derive from lineage restriction ofembryonic stem (ES) cells or induced pluripotent stem (iPS) cells. EScells are pluripotent stem cells derived from early embryos and can giverise to adult cells of all three germ layers. The iPS cells arepostnatal cells that are reprogrammed with transcription factors orsmall molecules to have phenotypic traits similar to ES cells and cangive rise to adult fates of all three germ layers with the caveat thatthey retain some molecular features in their chromatin reflecting thegerm layer from which the somatic cells derived. Multipotent or bipotentprogenitors and transit amplifying cells are precursors that havelimited (if any) ability to self-replicate and can give rise to 2 ormore adult fates. Committed progenitors are unipotent, have noself-replication capacity and give rise to only one adult cell type.

The cells from these types of stem cells/progenitors listed above andalso mature cells can successfully engraft as long as there are sources,ideally cellular sources, of multiple matrix metallo-proteinases (MMPs),both secreted and membrane-associated ones. MMPs are produced by allcell types, both immature and mature cells, but they vary in whichisoforms are produced and at what level of expression of particularMMPs. Representative secreted ones include MMP1, MMP2, MMP7 and MMP9.Representative membrane-associated ones include MMP14 and MMP15.Empirically it has been found that the highest production of secretedMMPs is by early lineage stage cells, stem cells and early progenitors.The biomaterials of the graft support the ability of both the epithelialand mesenchymal cells to produce these multiple forms of matrixmetallo-proteinases (MMPs) that remodel capsules around organs ortissues along with remodeling the subjacent tissue to the capsules andenabling migration of cells by means of dissolution of multiple forms ofextracellular matrix components.

The “rules” for engraftment are that there must be a source of MMPs,especially secreted isoforms, meaning that success occurs when

-   -   1) both the epithelial and mesenchymal cells are        stem/progenitors (so, both are cellular sources of MMPs)    -   2) the epithelial cells are stem progenitors and partnered with        mature mesenchymal cells (so the epithelial stem/progenitors are        the cellular source of MMPs)    -   3) the epithelial cells are mature cells that are partnered with        mesenchymal stem/progenitors (so the mesenchymal cells are the        cellular sources of MMPs)    -   4) theoretically, it is plausible to provide purified sources of        MMPs (i.e. cloned forms of MMPs) in the graft.

Matrix Metallo-proteinases (MMPs)

More generally, matrix metallo-proteinases (MMPS) are a large family ofzinc-dependent proteinases involved in breakdown and modulation ofextracellular matrix component and that are involved in implantation,invasion, angiogenesis, vascularization, and migration in normal andpathogenic processes. There are at least 24 isoforms that comprisematrixins, adamalysins, astacins, and serralysins. Their roles have beencharacterized in normal processes such as the implantation of theplacenta as well as in pathogenic ones such as invasion and metastasesof cancers. The studies described herein offer evidence for entirely newroles that contribute to engraftment, migration and integration oftransplanted cells. Stem/progenitors, both epithelial ones andmesenchymal ones, express multiple MMP isoforms that are especiallypotent in these roles. Maturation of the cells results in muting of theexpression of one or more of the potent stem/progenitor-cell-associatedMMPs and so diminishing the invasion and migration processes. Adultcells also express MMPs, primarily ones that are membrane bound(MT-MMPs), and being ones shown involved in plasticity processes but notthe wholesale engraftment and integration of cells into tissues. The netsum of this realization is that the graft biomaterials, backing andother conditions must be ones that optimize expression of the variousMMPs, such as the secreted MMPs, enabling the grafting and migrationprocesses to occur. Therefore, factors driving differentiation of thetransplanted cells will, in parallel, mute the complex MMP responses.This realization means that factors to be avoided include serum, solublesignals that drive differentiation (e.g. certain growth factors,cytokines and hormones); extracellular matrix components that drivedifferentiation (e.g. collagens, adhesion molecules, highly sulfatedglycosaminoglycans/proteoglycans); and mechanical forces contributingrigidity to the graft. In some embodiments, the one or more biomateriallayers comprise recombinant MMPs. In some embodiments, the one or morebiomaterial layers comprise cells engineered to express MMPs.

The term “mesenchymal cells” refers to cells derived from themesenchyme, including but not limited to mesenchymal stem cells whichare multipotent stromal cells and various subpopulations of mature andprogenitor mesenchymal cells of which there are at least two majorcategories:

Mature mesenchymal cells producing and surrounded by forms ofextracellular matrix that comprise fibrillar collagens (e.g. type I,III, V) and associated matrix components and bound signals (e.g. growthfactors/cytokines) that form a complex associated with cells that aretypically linear (string-like) cell populations. Nonlimiting examples ofsuch cells include stellate cells, tendon, stroma, and myofibroblasts.

Mature mesenchymal cells that produce and are surrounded by forms ofextracellular matrix that comprise network collagens (e.g. type IV, typeVI, VIII, X) and associated matrix molecules and bound signals (e.g.growth factors, cytokines) that together are associated with cellshaving more squamous or cuboidal or cobblestone morphologies.Non-limiting examples of such cells include endothelia andmyoepithelial.

The precursors to these mesenchymal cell types include but are notlimited to angioblasts which are multipotent and that can differentiateinto lineages of endothelia (the late stages of which are fenestratedendothelia) or stellate cells (the late stages of which aremyofibroblasts (stroma). The precursors also include mesenchymal stemcells (MSCs) which are multipotent cells and can differentiate intofibroblasts (stroma), osteoblasts (bone cells), chondrocytes (cartilagecells), myocytes (muscle cells) and adipocytes (fat cells).

The term “epithelial cell expansion” is correlated with the diameter ofa colony of epithelial cells that typically form colonies with cuboidalor cobblestone morphologies and with estimates of growth being thecomposite of the diameters of the cells of the colony. By contrast,estimates of growth of mesenchymal cell colonies are correlated with thedensity of the colony, since the mesenchymal cells are more migratoryand motile, and the colony density is a reflection of the net sum ofcells that remain within the colony boundaries.

The term “epithelial cells” refers to cells derived from the epithelium,specialized cells that provide diverse functions for the tissue and/orthe systemic needs of a host. They are recognized by their ability tomigrate as precursors or immature cells; with maturation, they becomestationary and form layers of squamous or cobblestone-like or columnarpolarized cells with apical, basal and lateral sides, and that are boundto each other by an assortment of junctions (connexins, tight junctions,adherens). Their expansion potential is indicated by the diameter of acolony (not by its density). The mature epithelial cells provide diversefunctions such as secretion of specialized products or of contributionsto metabolism (hepatocytes, cholangiocytes), detoxification(hepatocytes), production of enzymes (acinar cells), production ofendocrine factors (e.g. islets or other endocrine cells), electricalactivity (neuronal cells), and absorption (intestinal cells).

As used herein, the term “supportive” is used to describe cells whichare able to assist in the propagation of cells from another lineage orprovide assistance to neighboring cells through the production of“paracrine signals”, factors active in their effects on neighboringcells in terms of survival, expansion, migration, differentiation, andmaturation. For example, supportive mesenchymal cells may be defined bytheir ability to influence epithelial cells, optionally through thesecretion of matrix metallo-proteinases (MMPs) and/or one or moreother/or factors.

The term “lineage stage partners” refers herein to mesenchymal cellsthat are lineage stage appropriate for a given lineage stage ofepithelia and can support engraftment of the epithelial cells. For thehepatic or biliary tree stem cells, these are comprised of angioblasts(CD117+, CD133+, VEGFr+, CD31-negative) and their immediate descendants,precursors to endothelia (CD133+, VEGFr+, CD31+) and precursors tostellate cells (CD146+, ICAM-1+, alpha-smooth muscle actin+(ASMA),vitamin A-negative). The paracrine signaling between the epithelia andthe mesenchymal cells is also lineage-stage dependent, meaning that thespecific paracrine signals and the levels of them are distinct in early,intermediate and late stage cells. They can be mimicked, in part, by useof mesenchymal stem cells (MSCs), ones derived from bone marrow or fattissue. We refer to these collectively as early lineage stagemesenchymal cells (ELSMCs).

In some embodiments, the epithelial cells comprise biliary tree stemcells (BTSCs) and the mesenchymal cells comprise early-lineage-stagemesenchymal cells (ELSMCs). In some embodiments, the ELSMCs compriseangioblasts and their immediate descendants, precursors to endotheliacells, precursors to stellate cells, or combinations thereof. In someembodiments, the angioblasts express CD117, CD133, VEGFr, but do notexpress CD31. In some embodiments, the precursors to endothelia cellsexpress CD133, VEGFr, CD31 and Van Willebrand Factor. In someembodiments, the precursors to stellate cells express CD146, ICAM-1,alpha-smooth muscle actin (ASMA) and are negative for vitamin A.

The term “biliary tree stem cells” (BTSCs) refers to stem cells foundthroughout the intrahepatic and extrahepatic biliary tree and locatedwithin peribiliary glands (PBGs), both extramural and intramural, aswell as within the crypts of gallbladder villi and in Brunner's Glands.They have the ability to transition into committed hepatic and/orpancreatic progenitor cells. Thus far, at least 7 subpopulations of stemcell populations with overlapping traits and ranging from extremelyprimitive BTSCs to stem cell populations definable as hepatic orpancreatic stem cells have been identified in the intramural network,within the duct walls of the biliary tree.

Description of what is known for these stem cell subpopulations is givenbelow and outlined in FIG. 22. The most primitive ones are found in boththe extramural peribiliary glands—ones tethered to the surface of thebile ducts—and; the intramural peribiliary glands—ones found within thebile duct walls. The intramural peribiliary glands (PBGs) near to thefibromuscular layer in the centers of the bile duct walls can also beconsidered crypts (with parallels to intestinal crypts), niches in whichare found the most primitive stem cell populations. The largest numbersof the PBGs within the biliary tree network are found within thehepato-pancreatic common duct and within the large intrahepatic bileducts. No PBGs occur in the gallbladder, and instead the stem cellniches within the gallbladder are at the bottoms of the gall bladdervilli and that contain intermediate to late stage stem cell populationsthat are precursors to hepatic stem cells.

In some embodiments, the patch graft comprises BTSCs positive for atleast one marker selected from the group consisting of pluripotencygenes, such as OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2, and Bmi1, at leastone marker selected from the group consisting of endodermaltranscription factors, such as Sox9, Sox17, Pdx1, HNF4alpha and ONECUT2,at least one marker selected from the group consisting of surfacemarkers of stem/progenitors, such as EpCAM, LGR5, NCAM, one or moreisoforms of CD44, CXCR4, sodium iodide symporter (NIS), CD49 (integrinA6), CD29 (integrin B1), integrin B4. The BTSCs give rise to two stemcell subpopulations: hepatic stem cells (HpSCs) and pancreatic stemcells (PSCs), both having lower levels of expression of pluriotencygenes, similar levels of expression of surface markers ofstem/progenitors, and then markers defining HpSCs versus PSCs: forHpSCs, SOX9, SOX17, HNF4 alpha, ONECUT2, and constitutive expression ofalbumin; for PSCs, Pdx1, Ngn3, PTF1A, HNF1B, MUC6, PAX6, andconstitutive expression of insulin. The BTSCs, HpSCs and PSCs arenegative for markers of mature hepatic or mature pancreatic genes suchas expression of P450s, aquaporin, enzymes involved in bile production,digestive enzymes produced by mature acinar cells and regulatedexpression of albumin or regulated expression of insulin and of islethormones.

All of the BTSCs subpopulations express biomarkers that includeendodermal transcription factors for both liver and pancreas (e.g. SOX9,SOX17, PDX1), pluripotency genes (e.g. OCT4, SOX2, NANOG, SALL4,KLF4/KLF5, BMI-1); one or more of the hyaluronan receptor isoforms(standard and/or variant isoforms) of CD44; CXCR4; and cytokeratins 8and 18. Stem cell subpopulations within the biliary tree may include:

-   -   1: Cells found within Brunner's Glands (potentially another form        of BTSCs or a separate cell population—studies to define this        are still ongoing), located only in the submucosa of the        duodenum and nowhere else in the intestine. They express the        markers noted above and also Tra-160, Tra-181 and cytokeratin 7.        They are distinguishable from intestinal stem cells by their        traits.    -   2: Early stage Biliary Tree Stem Cells (BTSCs) that express        sodium iodide symporter (NIS) and CXCR4, OCT4, SOX2, NANOG, but        do not express LGR5 or EpCAM;    -   3. Intermediate stage of BTSCs that express less of NIS but gain        expression of LGR5 but not EpCAM;    -   4. Late stage BTSCs (the only BTSCs found in the gallbladder)        and also found in high numbers in the large intrahepatic bile        ducts and in the hepato-pancreatic common duct. They express        both LGR5 and EpCAM. These are precursors to hepatic stem cells        and to the pancreatic stem cells    -   5. Hepatic stem cells refers to stem cells found in the canals        of Hering, in PBGs of the large intrahepatic bile ductules, in        PBGs in the extrahepatic biliary tree; and in the PBGs of the        hepato-pancreatic common duct, but the highest numbers are those        at intrahepatic sites. The hepatic stem cells retain the ability        to self-replicate and to be multipotent. The biomarkers for        these cells include SOX9, SOX17, HNF-alpha, ITGB1 (CD29), ONECUT        2, SALL4, LGR5, CD44, epithelial cell adhesion molecule (EpCAM)        found in the cytoplasm and at the plasma membrane, neural cell        adhesion molecule (NCAM), negligible levels of constitutively        regulated (or no expression) of albumin, a complete absence of        alpha-fetoprotein (AFP), an absence of P450 A7, and an absence        of secretin receptor (SR). Hepatic stem cells and their        descendants, hepatoblasts, express cytokeratins 8, 18, and 19.    -   6. Pancreatic Stem cells are found in small numbers throughout        the biliary tree (even in the PBGs in the large intrahepatic        bile ducts) but are found in high numbers in PBGs of the        hepato-pancreatic common duct. They have the pluripotency genes        and expression for the other genes noted for all of the stem        cell populations, but they differ in no longer having SOX17.        Subpopulations of them that will lineage restrict to islets        express NGN3. They express EpCAM throughout the cells and at the        plasma membrane and express low, constitutively regulated (or        no) insulin. Maturation of them is correlated with increasing        insulin expression and its ability to be regulated by various        factors.

Intermediates in the lineage network refers to “transit amplifyingcells”, cells that can be bipotent (or multipotent), have considerableproliferative potential but demonstrate little (if any) trueself-replication, have low to moderate (or even no) pluripotency geneexpression, and express traits indicating commitment to an hepatic (e.g.albumin, alpha-fetoprotein) or a pancreatic (e.g. insulin, MUC6) fate.These include hepatoblasts (the network giving rise to liver) andpancreatic ductal progenitors (the network giving rise to pancreas).

As used herein, the term “pancreatic ductal progenitors” refers tobipotent cells found within pancreatic ductal glands (PDGs) within thepancreas and giving rise to acinar cells and islets. In our studies, wefind that they express SOX9, PDX1, HNF1f3, EpCAM, LGR5, ICAM-1, CD44,and subpopulations express NGN3 or MUC6.

As used herein, the term “hepatoblasts” refers to bipotent hepatic cellsthat can give rise to hepatocytic and cholangiocytic lineages and arefound in or adjacent to canals of Hering or in PBGs within the largeintrahepatic bile ducts. They have an extraordinary ability toproliferate (that is expand) but with less ability (if any) toself-replicate relative to that observed in hepatic stem cells. Thesecells are characterized by a biomarker profile that overlaps with but isdistinct from hepatic stem cells. They express SOX9, low (or evennegligible) levels of SOX17, high levels of LGR5, HNF4-alpha, and EpCAM,found primarily at the plasma membrane, and expressing P450A7,cytokeratin 7, secretin receptor, consistent and regulated expression ofalbumin in all hepatoblasts, high levels of alpha-fetoprotein (AFP),intercellular adhesion molecule (ICAM-1) but no expression of NCAM, andnegligible or no expression of pluripotency genes (e.g. SALL4, KL4/KLF5,OCT4, SOX2, NANOG).) and no expression of mature hepatic parenchymalmarkers (e.g. P450s such as P4503A).

As used herein the term “committed progenitor” refers to a unipotentprogenitor cell that gives rise to a single cell type, e.g. a committedhepatocytic progenitor cell. In some embodiments, they do not expresspluripotency genes. The committed hepatocytic progenitors are recognizedby expression of albumin, AFP, glycogen, ICAM-1, various enzymesinvolved with glycogen synthesis, and the gap junction gene, connexin28. These give rise to hepatocytes. A committed biliary (orcholangiocytic) progenitor gives rise to cholangiocytes and isrecognized by expression of EpCAM, cytokeratins 7 and 19, aquaporins,CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and membranepumps associated with production of bile. In some embodiments, acommitted islet progenitor expresses insulin, glucagon, and other islethormones albeit at low levels; with maturation the expression levels ofthe islet hormones increase but with particular cells expressingpreferentially certain hormones.

As used herein, the term “aggregates” refer to a plurality of cells thatare amassed together. The aggregates may vary in both size and shape ormay be substantially uniform in size and/or shape. The cell aggregatesused herein can be of various shapes, such as, for example, a sphere, acylinder (preferably with equal height and diameter), or rod-like amongothers. Although other shaped aggregates may be used, in one embodimentof the disclosure, it is generally preferable that the cell aggregatesbe spherical or cylindrical. The term “non-aggregated” refers toindividual, or single-celled, stem and/or progenitor cells. In someembodiments, the compositions provided herein can comprise substantiallyaggregated cells, substantially non-aggregated cells, or a mixturethereof.

The term “organoid” refers herein to a particular cellular aggregate ofdonor epithelial cells with mesenchymal cells that is self-assembled bysimple panning methods described herein. Organoids can be obtained frommixing of early stages of epithelia (ES cells, iPS cells, determinedstem cells, transit amplifying cells, progenitors) with early stages ofmesenchymal cells (angioblasts, precursors to endothelia, precursors tostellate cells).

Mixtures of adult epithelial cells with mature mesenchymal cells andchimeric mixtures of mature epithelial cells with early lineage stagemesenchymal cells (ELSMCs) do not usually generate organoids but can beused as mixtures of the cells in suspension in the graft biomaterials.If mature epithelia (e.g. hepatocytes, cholangiocytes, islets, acinarcells, enterocytes, etc.) are partnered with mature mesenchymal cells(e.g., endothelia, stellate cells, stromal cells, myofibroblasts), themixtures will not result in successful grafts but rather in ones thatpersist at the surface of the organs or tissues. It is hypothesized thatthis is because they express plasma-membrane-associated MMPs but minimallevels of secreted MMPs. If chimeric mixtures are used (e.g. maturehepatocytes with angioblasts), then engraftment does occur, since thereis a source of secreted MMPs that enable engraftment and migration ofthe cells.

Protocols for Establishing Organoids.

According to one embodiment disclosed herein, organoids, of biliary treestem cells (BTSCs) and early lineage stage mesenchymal cells (ELMCs)proved the most successful method of incorporating cells in the grafts.It is disclosed herein that BTSCs and ELMCs can self-select intoorganoids by panning to eliminate the mature stellate/stromal cells, andthis a proved more efficient and effective in establishing lineage-stageappropriate epithelial-mesenchymal partners for the grafts. In anotheraspect, this disclosure provides a methods of forming organoids byculturing a first type of cells with a second type of cells, wherein thesecond type of cells is a stage appropriate lineage partner of the firsttype of cells, removing mature cells that attach to the culture dish bypanning, and recovering the self-assembled organoids from the suspensionof the culture. The first type of cells may be epithelial stem cells orcommitted epithelial cells. The second type of cells may be cells of themesenchymal lineage, mesenchymal stem cells, or early lineage stagemesenchymal cells.

In some embodiments, the mesenchymal cells are supportive mesenchymalcells. In some embodiments, the organoids are formed after culturing onlow attachment dishes and under serum-free, wholly defined conditionstailored to the lineage stage(s) of the aggregated cells in suspension.

In some embodiments, the mixture of epithelial cells and mesenchymalcells is produced by depleting cell suspensions of mature mesenchymalcells, optionally, by repeated panning procedures to remove cells thatattach within from about 15 minutes to about 30 minutes on tissueculture dishes or surfaces at 37° C. As used herein, the term“produced”, its equivalents (e.g. producing, produce, etc.) are usedinterchangeable with “generated” or “formed” and their equivalents whenreferring to the method steps that bring the organoid of the instantdisclosure into existence. Multiple rounds (e.g. 4-5) of such a panningprocess enriches the cell suspension for the earlier lineage stagecells. Then the cell suspension is transferred to low attachment dishesand again in serum-free medium, one designed for the early lineage stagecells, and left for some hours or even overnight in an incubator at 37°C. In some embodiments, a culturing of the remaining cell suspensions isperformed on low-attachment dishes and in a serum-free medium until aplurality of organoids is formed by self-assembly of epithelial cellsand mesenchymal cells. In some embodiments, the serum-free mediumcomprises a basal medium (with no copper, low calcium (0.3 mM), 1 nMselenium, 0.1% bovine serum albumin (purified, fatty-acid-free; fractionV), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg/mltransferrin/Fe, 5 μg/ml insulin, and a mixture of purified free fattyacids that are presented complexed with fatty acid free highly purifiedalbumin. In some embodiments, in which the serum-free medium furthercomprises 10 μg/ml high density lipoprotein. Additional, serum-freemedium compositions are described elsewhere herein.

In some embodiments, the plurality of organoids comprises BTSCs positivefor:

-   -   at least one marker selected from the group of pluripotency        genes consisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2 and        Bmi1,    -   at least one marker selected from the group of endodermal        transcription factors consisting of Sox9, Sox17, Pdx1,        HNF4alpha, HNFB1 and ONECUT2,    -   at least one marker selected from the group of surface markers        associated with stem/progenitors consisting of EpCAM, NCAM,        LGR5, one or more isoforms of CD44, CXCR4, sodium iodide        symporter (NIS), CD49 (integrin A6), CD29 (integrin B1) and        integrin B4;        -   wherein the BTSCs are negative for markers of mature hepatic            or pancreatic cells, including P450s, aquaporin, enzymes            involved in bile production, amylase and digestive enzymes.

In some embodiments, the culturing of the remaining cell suspensions isperformed on low-attachment dishes and in a serum-free medium until aplurality of organoids is formed by self-assembly of cells remaining inthe cell suspension. In some embodiments, the plurality of organoids isformed after about 2 hours, after about 4 hours, after about 6 hours,after about 8 hours, after about 10 hours, after about 12 hours, afterabout 14 hours, after about 16 hours, after about 18 hours, after about20 hours, after about 22 hours, or after about 24 hours.

Cell Culture Conditions.

The term “culture” or “cell culture” means the maintenance of cells inan artificial, in vitro environment. A “cell culture system” is usedherein to refer to culture conditions in which a population of cells maybe grown ex vivo (outside of the body).

The term “basal media” refers to buffers used for cell culture and arecomprised of amino acids, sugars, lipids, vitamins, minerals, salts,trace elements, and various nutrients in compositions that mimic thechemical constituents of interstitial fluid around cells.

“Culture medium” is used herein to refer to a nutrient solution for theculturing, growth, or proliferation of cells. Culture medium may becharacterized by functional properties such as, but not limited to, theability to maintain cells in a particular state (e.g. a pluripotentstate, a proliferative state, quiescent state, etc.), to mature cells—insome instances, specifically, to promote the differentiation ofprogenitor cells into cells of a particular lineage. Non-limitingexamples of culture media are serum supplemented media (SSM) being anybasal medium supplemented with serum at levels that are typically about10% to about 20%. The serum can be autologous (the same species as thecells) or, more commonly, serum from animals that are routinelyslaughtered for commercial purposes (e.g. chickens, cows, pigs, etc.).

For the grafting technologies, conditions are used to maintain the cellsas stem cells or early progenitor cells; there is an avoidance of serumor any of the typical supplements that might drive the cells down adifferentiation pathway and towards a mature cell fate. In addition tothe customary basal media, various nutritional supplements, lipids(mixture of free fatty acids complexed with albumin and carriermolecules such as high density lipoprotein). Only two hormone/growthfactors are added: insulin needed for carbohydrate metabolism, andtransferrin, needed as a Fe carrier for the polymerases.

“Kubota's Medium” as used herein refers to any basal medium containingno copper, low calcium (<0.5 mM), selenium, zinc, insulin,transferrin/Fe, a mix of free fatty acids bound to purified albumin and,optionally, also high density lipoprotein (HDL). In some embodiments,Kubota's Medium comprises any basal medium (e.g., RPMI 1640 or DMEM-F12)with no copper, low calcium (e.g., 0.3 mM), ˜10⁻⁹ M selenium, ˜0.1%bovine serum albumin or human serum albumin (highly purified and fattyacid free), ˜4.5 mM nicotinamide, ˜0.1 nM zinc sulfate heptahydrate,˜10⁻⁸ M hydrocortisone (optional component used for hepatic but notpancreatic precursors), ˜5 μg/ml transferrin/Fe, ˜5 μg/ml insulin, ˜10μg/ml high density lipoprotein, and a mixture of purified free fattyacids that are added after binding them to purified serum albumin. Thefree fatty acid mixture consists of ˜100 mM each of palmitic acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and stearicacid. Non-limiting, exemplary methods for the preparation of this mediahave been published elsewhere, e.g., Kubota H, Reid L M, Proc. Nat.Acad. Scien. (USA) 2000; 97:12132-12137, the disclosure of which isincorporated herein in its entirety by reference.

Non-limiting examples of defined media to differentiate include thehormonally-defined media (HDM) used for differentiation of endodermalstem cells to adult fates. Supplements can be added to Kubota's Mediumto generate a serum-free, hormonally defined medium (HDM) that willfacilitate differentiation of the normal hepatic or biliary tree stemcells to specific adult fates. Non-limiting examples of defined media todifferentiate endodermal stem cells to adult fates include onessupplemented with calcium to achieve at or above 0.6 mM concentration, 1nM tri-iodothyronine (T3), 10⁻¹² M copper, 10 nM of hydrocortisone and20 ng/ml of basic fibroblast growth factor (bFGF). The medium conditionsover and above these needed to selectively yield hepatocytes (HDM-H)versus cholangiocytes (HDM-C) versus pancreatic islets (HDM-P) are:

-   -   HDM-H: supplementation further with 7 μg/L glucagon, 2 g/L        galactose, 10 ng/ml epidermal growth factor (EGF) and 20 ng/ml        hepatocyte growth factor (HGF);    -   HDM-C: supplementation further with 20 ng/ml vascular        endothelial cell growth factor (VEGF) and 10 ng/ml HGF; and    -   HDM-P: prepared without glucocorticoids and further supplemented        with 1% B27, 0.1 mM ascorbic acid, 0.25 μM cyclopamine, 1 μM        retinoic acid, 20 ng/ml of FGF-7 for 4 days, then changed with        one supplemented with 50 ng/ml exendin-4 and 20 ng/ml of HGF for        6 more days of induction.

The HDM provided herein can be supplements with additional growthfactors including, but not limited to, Wnt ligands, R-spondins,epidermal growth factors (EGFs), fibroblast growth factors (FGFs),hepatocyte growth factors (HGFs), insulin-like growth factors (IGFs),transforming growth factors (TGFs), nerve growth factors (NGFs),neurotrophic factors, various interleukins, leukemia inhibitory factors(LIFs), vascular endothelial cell growth factors (VEGFs),platelet-derived growth factors (PDGFs), stem cell factors (SCFs),colony stimulating factors (CSFs), GM-CSFs, erythropoietin,thrombopoietin, heparin binding growth factors, IGF binding proteins,and/or to placental growth factors.

The HDM provided herein can be supplemented with cytokines including,but not limited to interleukins, lymphokines, monokines, colonystimulating factors, chemokines, interferons and tumor necrosis factor(TNF).

In some embodiments the medium may be a “seeding medium” used to presentor introduce cells into a given environment. In other embodiments, themedium may be a “differentiation medium” used to facilitate thedifferentiation of cells. Such media are comprised of a “basal medium”,a mixture of nutrients, minerals, amino acids, sugars, lipids, and traceelements and supplemented either with serum (serum supplemented media orSSM) or with a defined mix of purified hormones, growth factors andnutrients, a hormonally defined medium (HDM), and used for survival,maintenance or differentiation of cells ex vivo. As used herein, “HDM-H”is an HDM used in combination with substrata of type IV collagen andlaminin to drive the differentiation of endodermal stem/progenitors tomature hepatocytes. HDM-C is an HDM used with substrata of type Icollagen and fibronectin to drive the cells to mature cholangiocytes.

As used herein, the HDM can also be used with substrata of purifiedextracellular matrix components or extracts enriched in extracellularmatrix, matrix substrata that facilitate differentiation to specificfates. An example is the use of “HDM-H” in combination with substrata ofpurified type IV collagen and laminin to drive the differentiation ofendodermal stem/progenitors to mature hepatocytes. HDM-C is an HDM thatcan be used with substrata of type I collagen and fibronectin to drivethe cells to mature cholangiocytes.

The HDM can be used also in combination with extracellular matrixextracts resulting from decellularization processes such as those usedto isolate biomatrix scaffold. Further details on HDM can be found in WO2012/003463, U.S. Pat. Nos. 9,102,913, 8,802,081 and WO 2012003450,incorporated herein.

Basal media are buffers used for cell culture and are comprised of aminoacids, sugars, lipids, vitamins, minerals, salts, trace elements, andvarious nutrients in compositions that mimic the chemical constituentsof interstitial fluid around cells. In addition, cell culture media areusually comprised of basal media supplemented with a small percentage(typically 2-10%) serum. For the grafting technologies, conditions areused to maintain the cells as stem cells or early progenitor cells andso there is an avoidance of serum or any of the typical supplements thatmight drive the cells down a differentiation pathway and towards amature cell fate. In addition to the customary basal media, variousnutritional supplements, lipids (mixture of free fatty acids complexedwith albumin and carrier molecules such as high density lipoprotein).Only two hormone/growth factors are added: insulin needed forcarbohydrate metabolism, and transferrin, needed as a Fe carrier for thepolymerases. Kubota's medium, a serum-free medium designed forendodermal stem/progenitors is comprised of a basal medium supplementedwith zinc, selenium, insulin, transferrin, lipids but no cytokines orgrowth factors. Other growth factors and cytokines and especially serumare to be avoided since they will induce differentiation of the donorcells and, thereby, minimize the production of MMPs, required for theengraftment and migration processes.

In some embodiments, the conditions of these patch grafts are,therefore, counter to the routine use of media supplemented with a smallpercentage (typically 2-10%) serum. Serum has long been added to providerequisite signaling molecules (hormones, growth factors, cytokines)needed to drive a biological process (e.g. proliferation,differentiation), but in these strategies for patch grafts serum is tobe avoided to enable engraftment to occur. In some embodiments, serum isnot included to avoid differentiation of the cells and/or avoidinactivating or muting secreted forms of MMPs.

In some embodiments, the serum-free medium comprises a basal medium(with no copper, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serumalbumin (purified, fatty-acid-free; fraction V), 4.5 mM nicotinamide,0.1 nM zinc sulfate heptahydrate, 5 μg/ml transferrin/Fe, 5 μg/mlinsulin, and a mixture of purified free fatty acids that are presentedcomplexed with fatty acid free highly purified albumin. In someembodiments, the serum-free medium further comprises 10 μg/ml highdensity lipoprotein.

Methods of Using the Patch Graft Compositions

In one aspect, the present disclosure relates method of engrafting cellsinto a solid organ of a subject in need thereof, comprising:

-   -   contacting a patch graft onto a solid organ,        -   the patch comprising a mixture of epithelial cells and            mesenchymal cells incorporated into a biomaterial having a            first viscoelasticity property, in which the biomaterial            promotes an engraftment of at least a portion of said            epithelial cells, mesenchymal cells, or both among the cells            of the solid organ;    -   demonstrating that at least a portion of said epithelial cells,        mesenchymal cells, or both have engrafted among the cells of the        solid organ.

In some embodiments, demonstrating comprises measuring a level of asecretion from the solid organ, or a metabolic effect of the solidorgan, in a biological sample obtained from the subject to demonstratethat at least a portion of said epithelial cells have engrafted amongthe cells of the solid organ.

In another aspect, the present disclosure relates to a method ofengrafting cells into a solid organ of a subject in need thereof,comprising:

-   -   contacting a patch graft onto a solid organ,        -   the patch comprising a mixture of epithelial cells and            mesenchymal cells incorporated into a hydrogel layer having            a first viscoelasticity property, in which the hydrogel            promotes a migration of at least a portion of said            epithelial cells, mesenchymal cells, or both from the patch            through an outer surface of the solid organ,    -   demonstrating that at least a portion of said epithelial cells,        mesenchymal cells, or both have migrated through an outer        surface of the solid organ.

As used herein, the term “engraftment” refers to incorporating cellsinto a tissue or organ. Alternatively, the terms grafting,transplantation, engraftation, ingraftation, or ingraftment may be usedinterchangeably with engraftment.

The term “migrate” refers to cellular movement from site to anotherwithin a tissue or organ.

The term “integration” refers to that the cells combine with the cellsof the host organ or tissue and become a part of the organ or tissue,but do not fuse with host cells.

In some embodiments, at least a portion of the mixture of epithelialcells and mesenchymal cells migrates over the substantial width of thesolid organ and distributes throughout the solid organ. In someembodiments, the patch graft further comprises a backing that promotes amigration of at least a portion of the mixture of epithelial cells andmesenchymal cells towards the solid organ.

In some embodiments, the epithelial cells are early lineage stageepithelia cells (ELSEs), and the mesenchymal cells are early lineagestage mesenchymal cells (ELSMCs).

In some embodiments, the ELSMCs comprises angioblasts, precursors ofendothelia, stellate cells, or a combination thereof. In someembodiments, the ELSEs and/or the ELSMCs are derived from embryonic stem(ES) cells or from induced pluripotent stem cells (iPS). In someembodiments, the epithelial cells are mature and the mesenchymal cellsare ELSMCs. In some embodiments, at least one of the two categories ofdonor cells may be a stem/progenitor enabling capable of the productionof MMPs. In a preferred embodiment, the MMPs are secreted isoforms ofMMPs.

The terms “tissue” is used herein to refer to tissue of a living ordeceased organism or any tissue derived from or designed to mimic aliving or deceased organism. The tissue may be healthy, diseased,injured by trauma, damaged and/or have genetic mutations. The term“natural tissue” or “biological tissue” and variations thereof as usedherein refer to the biological tissue as it exists in its natural or ina state unmodified from when it was derived from an organism. A“micro-organ” refers to a segment of “bioengineered tissue” that mimics“natural tissue.”

The biological tissue may include any single tissue (e.g., a collectionof cells that may be interconnected) or a group of tissues making up anorgan or part or region of the body of an organism. The tissue maycomprise a homogeneous cellular material or it may be a compositestructure such as that found in regions of the body including the thoraxwhich for instance can include lung tissue, skeletal tissue, and/ormuscle tissue. Exemplary tissues include, but are not limited to thosederived from liver, pancreas, biliary tree, lung, intestine, thyroid,thymus, bladder, kidneys, prostate, uterus, breast, skin, brain, spinalcord, blood vessels (e.g. aorta, iliac vein,), heart, muscle, includingany combination thereof.

In some embodiments, the mixture of stem/progenitor cells andmesenchymal cells migrates over much of or if not the entire width ofthe organ and distributes uniformly throughout the organ. In someembodiments, the solid organ is an endodermal organ. In someembodiments, the solid organ is an endodermal organ comprising liver,pancreas, intestine, lung, bile duct, thymus, thyroid, parathyroid andthe urogenital sinus region of the prostate and vagina. In someembodiments, the endodermal organ comprises liver, and engraftmentinvolves a remodeling of Glisson's Capsules.

As used herein, the term “remodeling” refers to histological changes intissue initiated by the engraftment and caused, in part, by the secretedMMPs. For example, in some embodiments, the engraftment processesdisclosed herein results in remodeling of the Glisson Capsule and of thehost tissue near to the graft. The remodeling of tissue is transient andreverts to normal histology after the cells are fully integrated intothe host organ/tissue. Remodeling can be visualized by multiple stainssuch as trichrome staining that identifies extracellular matrixcomponents. These are complemented by staining with H&E.

In some embodiments, the present disclosure provides methods whichfurther gives rise to a combination of (i) engrafted epithelial cellsand mesenchymal cells and (ii) host cells. In some embodiments, themethods of the present disclosure gives rise to functional hepaticparenchymal cells. In some embodiments, the parenchymal cells comprisehepatocytes and cholangiocytes.

In some embodiments, the patch includes a backing positioned over thehydrogel containing the mixture of stem/progenitor cells and mesenchymalcells. In some embodiments, the backing is used to tether the hydrogellayer to the target organ or site. In some embodiments, the endodermalorgan comprises pancreas, and engraftment involves a remodeling ofpancreatic capsules and pancreatic tissue near to the graft site. Insome embodiments, the methods of the present disclosure gives rise tofunctional pancreatic cells. In some embodiments, the functionalpancreatic cells comprise acinar cells and islets.

In some embodiments, demonstrating comprises measuring a parameter or achange in same, which indicates a physiological effect in the subjectresulting from the migrated cells.

In another aspect, the present disclosure relates to a method ofintroducing, restoring, increasing, or improving functionality of adiseased, impaired, or malfunctioning solid organ of a subject,comprising contacting the diseased, impaired, or malfunctioning solidorgan with a patch graft comprising a mixture of epithelial cells andmesenchymal cells under conditions that promote engraftment of theepithelial cells and mesenchymal cells; demonstrating an introduction,restoration, increase, or improvement of a functionality in thediseased, impaired, or malfunctioning solid organ.

In some embodiments, a portion of the mixture of stem/progenitor cellsand mesenchymal cells exists in combination with the cells of the targetorgan. In some embodiments, the patch graft used in the method ofrestoring organ function includes a coating that inhibits adhesion ofthe patch graft to organs and tissues in the vicinity of the patchgraft.

In some embodiments, demonstrating comprises measuring in a biologicalsample obtained from the subject a level of a secretion or metabolicproduct or effect. In some embodiments, the methods of the presentdisclosure further comprises demonstrating that a least a portion of themixture of epithelial cells and mesenchymal cells has distributed amongthe cells of the host organ. In some embodiments, an exposed surface ofthe patch graft includes a coating that inhibits adhesion of the patchgraft to organs and tissues in the vicinity of the patch graft. In someembodiments, the solid organ comprises an endodermal organ. In someembodiments, the endodermal organ comprises liver, pancreas, intestine,lung, bile duct, thymus, thyroid, parathyroid or the regions from theurogenital sinus of the prostate or vagina. In some embodiments, thesolid organ comprises a pancreas and in which an increased level of thesecretion of at least one of insulin, c-peptide glucagon, somatostatin,or pancreatic polypeptide is measured.

In some embodiments the solid organ comprises a pancreas and in which areduced blood sugar level is measured. In some embodiments, the solidorgan comprises a pancreas and in which increased glucose tolerance isdemonstrated. In some embodiments, the solid organ comprises a pancreasand in which increased levels of a digestive enzyme or bicarbonate fluidis demonstrated. In some embodiments, the digestive enzyme comprisesamylase, lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase,or elastase. In some embodiments, the solid organ comprises a pancreasand in which increased levels of a product from a digestive enzymesecreted by the pancreas is measured. In some embodiments, the digestiveenzyme comprises amylase, lipase, peptidase, ribonuclease,deoxyribonuclease, gelatinase, or elastase. In some embodiments, thesolid organ comprises a pancreas and in which improved digestion isdemonstrated.

In some embodiments, the solid organ comprises liver, and in which asecretion comprises urea, bile acids, phospholipids, lipoproteins,bilirubin, bicarbonate-rich fluids, blood-clotting factors, orcombinations thereof.

In some embodiments, the solid organ comprises liver, and in which ametabolic effect is a reduced level of one or more of cholesterol, bloodsugar, alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactatedehydrogenase. In some embodiments, a metabolic effect is a decrease inlevels of tyrosine or alpha-fetoprotein.

In another aspect, the present disclosure relates to a method oftreating a subject diagnosed with a pathological condition attributableat least in part to having a diseased, impaired, or malfunctioning solidorgan, comprising

-   -   (i) contacting the diseased, impaired, or malfunctioning solid        organ with a patch graft comprising a mixture of epithelial        cells and mesenchymal cells,    -   (ii) allowing the epithelial cells and mesenchymal cells to        migrate into and distribute among the cells of the host solid        organ, and    -   (iii) demonstrating that a negative effect of said diseased,        impaired, or malfunctioning solid organ has been alleviated in        the treated subject.

In some embodiments, demonstrating comprises measuring in a biologicalsample obtained from the subject a level of a secretion or a metabolicproduct or effect. In some embodiments, the migration and distributionsteps lead to an alleviation of the disease, impairment, or malfunction.

In some embodiments, the solid organ is an endodermal organ. In someembodiments, the endodermal organ comprises liver, pancreas, intestine,lung, bile duct, thymus, thyroid, parathyroid, and the urogenital sinusregions of the prostate or vagina. In some embodiments, the endodermalorgan is pancreas and in which the subject suffers from diabetes. Insome embodiments, increased levels of at least one of insulin,c-peptide, glucagon, somatostatin, or pancreatic polypeptide ismeasured. In some embodiments, reduced blood sugar levels aredemonstrated. In some embodiments, increased glucose tolerance isdemonstrated.

As used herein, the term “subject” and “patient” are usedinterchangeably and are intended to mean any animal. In someembodiments, the subject may be a mammal. In some embodiments, themammal is bovine, equine, porcine, canine, feline, simian, murine,human, or rat. In some embodiments, the subject is a human. In someembodiments, the subject comprises a mammal. In some embodiments, themammal is human.

As used herein, “treating” or “treatment” of a disease in a subjectrefers to (1) preventing the symptoms or disease from occurring in asubject that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of the present technology, beneficial or desired resultscan include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of acondition (including a disease), stabilized (i.e., not worsening) stateof a condition (including disease), delay or slowing of condition(including disease), progression, amelioration or palliation of thecondition (including disease), states and remission (whether partial ortotal), whether detectable or undetectable.

Also provided herein are methods of treating a subject with a liverdisease or disorder, the methods comprising, consisting of, orconsisting essentially contacting the subject's liver with a patch graftcomprising multiple layers including, at least: a first layer ofhydrogel comprising epithelial cells and mesenchymal cells; a secondlayer of hydrogel; a third layer comprising a biocompatible,biodegradable backing; and optionally a fourth layer of hydrogel. Insome embodiments of the methods, the liver disease or disorder is liverfibrosis, liver cirrhosis, hemochromatosis, liver cancer, biliaryatresia, nonalcoholic fatty liver disease, hepatitis, viral hepatitis,autoimmune hepatitis, fascioliasis, alcoholic liver disease, alpha1-antitrypsin deficiency, glycogen storage disease type II,transthyretin-related hereditary amyloidoisis, Gilbert's syndrome,primary biliary cirrhosis, primary sclerosing cholangitis, Budd-Chiarisyndrome, liver trauma, or Wilson disease.

In other aspects, provided herein are methods of treating a subject witha disease or disorder of the pancreas, the methods comprising,consisting of, or consisting essentially of contacting the subject'spancreas with a patch graft comprising multiple layers including, atleast: a first layer of hydrogel comprising epithelial cells andmesenchymal cells; a second layer of hydrogel; a third layer comprisinga biocompatible, biodegradable backing; and optionally a fourth layer ofhydrogel. In some embodiments of the methods, the disease or disorder ofthe pancreas is diabetes mellitus, exocrine pancreatic insufficiency,pancreatitis, pancreatic cancer, sphincter of Oddi dysfunction, cysticfibrosis, pancreas divisum, annular pancreas, pancreatic trauma, or hemosuccus pancreaticus.

In other aspects, provided herein are methods of treating a subject witha gastrointestinal disease or disorder, the method comprising,consisting of, or consisting essentially of contacting one or more ofthe subject's intestines with a patch graft comprising multiple layersincluding, at least: a first layer of hydrogel comprising epithelialcells and mesenchymal cells; a second layer of hydrogel; a third layercomprising a biocompatible, biodegradable backing; and optionally afourth layer of hydrogel. In some embodiments, the gastrointestinaldisease or disorder is gastroenteritis, gastrointestinal cancer,ileitis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, irritable bowel syndrome, peptic ulcer disease, celiac disease,fibrosis, angiodysplasia, Hirschsprung's disease, pseudomembranouscolitis, or gastrointestinal trauma.

Modes of Carrying Out the Disclosure

The patch graft composition provided herein is directed to directgrafting of cells into the solid organ. The method is safe, avoidsemboli and ectopic cell distribution, and optimizes cell numberengraftment and distribution into and throughout the tissue.

Aspects of the disclosure relate to compositions and methods forengrafting cells into an organ. Efforts to transplant cells from solidorgans into internal organs typically made use either of directinjection or by delivering cells via a vascular route. Lanzoni, G. etal. Stem Cells 31, 2047-2060 (2013). These methods of transplantationresult in small numbers of cells being transplanted, and in risks ofemboli that can be life threatening. Transplantation is improved if thecells are delivered by “injection grafting” in which the cells aresuspended in hyaluronans and then co-injected with a trigger (PEGDA)that causes the hyaluronan to gel in situ. Injection graftingmethodologies provide a strategy for localizing cells to a specificsite, albeit in small numbers, typically 10⁶-10⁷ cells per injectionsite. This strategy eliminates or minimizes ectopic cellulardistribution and optimizes the integration of the cells in the site.However, if mature functional cells are used, they may be highlyimmunogenic, necessitating long-term immunosuppression.

To address some of these hurdles and concerns, the challenges areovercome by “patch grafting” strategies described herein. In someembodiments, “bandaid-like” grafts are tethered surgically to thesurface of an organ or tissue; the conditions of the graft are such thatthe cells engraft fully into the site, migrate throughout theorgan/tissue, and then mature into relevant adult cell types. Thepotential for transplantation of large numbers of cells (e.g. >10⁸cells) is dictated entirely by the size of the patch, the number ofcells within the graft, and the source of multiple forms of MMPs,ideally cellular sources of the MMPs. Moreover, in some embodiments theuse of organoids facilitates the ability to stockpile donor cells giventhe ease by which the organoids can be cryopreserved under whollydefined, serum-free conditions.

The patch graft composition provided herein is directed to grafting ofcells into the solid organ. The method is safe, avoids emboli andectopic cell distribution, and optimizes cell number engraftment anddistribution into and throughout the tissue.

The present disclosure provides a novel method of transplantation ofcells into solid organs or tissues, including internal organs, anddemonstrated herein with studies on liver. The method is safe, avoidsemboli and ectopic cell distribution, and optimizes rapid engraftmentand distribution of large numbers of cells throughout the host tissue.Within a week, there was engraftment of all of the donor cells(≥10^(8th) in pigs; ≥10^(6th) in mice) throughout significant portionsof the liver followed by migration and integration of cells atsignificant distances from graft sites. Maturation to mature cell typesoccurred throughout regions of engrafted cells by 2 weeks, followed byrestoration of Glisson's capsules and of tissue histology andarchitecture by 3 weeks. Importantly, patch grafts were able to rescueanimals from a disease state, demonstrated here with patch grafts inmice models of type I tyrosinemia.

The strategies for transplantation of cells onto solid organs disclosedherein are radically different compared to those known in the art. Themethods of transplantation of cells onto solid organs disclosed hereinmay in some embodiments involve placing grafts directly onto the surfaceof the target site/organ and using graft biomaterials designed to enabledonor cells to engraft and migrate into the tissue. This parallelsstrategies of cell therapies for skin, but requires modifications forinternal tissues, such as abdominal internal organs, to account formechanical effects, abrasion or compression of organs near to eachother, and with recognition of the unique fluid microenvironments aroundspecific organs.

The inventors of the present disclosure demonstrated herein is someexemplary embodiments the strategy with grafts of biliary tree stemcells (BTSCs), which are determined endodermal stem/progenitors that areprecursors to both liver and pancreas. In some embodiments, the BTSCswere transplanted by patch grafting onto the surface of the liver. Insome embodiments, the BTSCs were transplanted by patch grafting onto thesurface of the pancreas.

The animal models used in some embodiments of the present disclosurecomprise mice (Mus musculus) and swine (Sus scrofa domestic)transplanted with donor cells with a transgene coupled to a fluoroprobe,green fluorescent protein (GFP), linked to a histone (H2-B) locus, thusproviding a nuclear biomarker. In some embodiments, the methods of thepresent disclosure used a murine model, NOD-Rag1−/−IL2RgammaC-null(NRG), that is immunocompromised and also genetically (using Crispr/Cas9technology) made deficient in fumaryl aceto-acetate hydrolase (FAH), akey enzyme in the tyrosine metabolic pathway. Its loss results in type Ityrosinemia. Mice and pigs are major animal species in translationalresearch and are used increasingly as alternatives to non-human primatesin preclinical studies.

The inventors of the present disclosure have previously reported thatengraftment required co-transplantation of epithelial cells with theirlineage-stage-appropriate mesenchymal cell partners. Turner, R., et al.Successful Transplantation of Human Hepatic Stem Cells With RestrictedLocalization to Liver Using Hyaluronan Grafts. Hepatology 57, 775-784(2013). For hepatic and biliary tree stem cells, these mesenchymal cellsare comprised of angioblasts (CD117+, CD133+, VEGFr+, CD31-negative) andtheir immediate descendants, precursors to endothelia (CD133+, VEGFr+,CD31+, Van Willebrand Factor+) and precursors to stellate cells (CD146+,ICAM-1+, alpha-smooth muscle actin+(ASMA), vitamin A-negative). We referto these collectively as early-lineage-stage-mesenchymal-cells (ELSMCs).

In some embodiments, matching epithelial and mesenchymal cell partnersmay be isolated by using multiparametric flow cytometry to determine theratios of lineage stage partners of epithelial and mesenchymal cells incell suspensions and then used those ratios within grafts usingimmuno-selected cells. In some preferable embodiments, it may be moreefficient to deplete cell suspensions of mature mesenchymal cells byrepeated panning procedures (see methods) followed by culturing theremaining cell suspensions on low attachment dishes and in serum-freeKubota's Medium for ˜6-8 hours. Organoids self-assembled with eachaggregate containing approximately 50-100 cells. Marker analysesindicated partnering of BTSCs with ELSMCs (FIGS. 1A-1D). As summarizedin FIG. 1A, the BTSCs/ELMCs were used immediately or were cryopreservedunder defined conditions and thawed as needed for grafts. Organoids ofBTSCs/ELSMCs were characterized using immunofluorescence (IF), qRT-PCRand RNA-seq and shown to express classic traits of BTSCs (FIGS. 1A-1D)and of ELSMCs (data not shown). BTSCs in the organoids expressed lowlevels of pluripotency genes (e.g. OCT4, SOX2) and endodermal stem cellgenes (e.g. EpCAM, SOX 9, SOX17, PDX1, LGR5, CXCR4, MAFA, NGN3 and NIS),but did not express mature hepatic or pancreatic genes. RepresentativeqRT-PCR assays confirmed these findings from cells prior totransplantation (FIG. 1D). Immunohistological (IHC) assays indicatedthat more primitive cells (e.g. ones expressing the highest levels ofpluripotency genes) were distributed to the interiors of the organoidsand later maturational lineage stages were at the perimeters (FIG. 1C).The results shown in FIGS. 1A-1D are exemplary embodiments for formationof porcine organoids. In some embodiments, organoids may similarly beformed from any mammal. In some embodiments, the organoids are formedfrom mice, or preferably from human cells.

Patch grafts were secured to the liver surface by sutures or surgicalglue as shown in FIGS. 2A-2E. The graft composition involved the use ofthiol-modified hyaluronans (HA) hydrogels prepared with preciseconcentrations of HA and PEGDA to achieve defined levels of stiffness asdetermined rheologically and expressed as the dynamic shear modulus (G*)(FIG. 2C). Donor cells were embedded into a soft HA layer (less than 100Pa) and placed against the liver surface, covered with the backingimpregnated with more rigid HA (˜700 Pa), and the graft sutured or gluedto the liver surface at the corners of the silk patch. The softhydrogels into which donor cells were placed maintained stemness traitsessential for production of matrix metallo-proteinases (MMPs) needed forengraftment. The silk backing impregnated with HA served as a barrier tomigration in directions other than towards the target tissue. An HAhydrogel with a rigidity of ˜200-300 Pa enabling painting or coating theoutside surface of grafts, was used at the time of the surgery andserved to minimize adhesions with surrounding tissues.

The only variant of patch grafting attempted and then abandoned wasafter sharp surgical removal of the capsule. Hemorrhaging was excessive,obviating future use in hosts with altered hemostasis associated withhepatic failure or even in normal hosts given the adverse influences ofserum on donor cells. Without such efforts to alter the organ capsules,patch grafts proved facile for surgical procedures.

A number of backings were tested with a focus on ones already usedclinically in abdominal surgeries as shown in Table 1 and 2 below. Onlythe SERI Silk Surgical Scaffolds (Sofregen, Medford, Mass.), did notcause problems. The problems of other backings (Table 1, FIGS. 12A-12E)included fragility (e.g. Seprafilm, Reglyde); induction of necrosis orfibrosis and significant levels of adhesions (e.g. Surgisis, Vetrix);and severe adhesion formations with a filamentous sponge version madefrom reconstituted silk protein or any of the backings supplemented withcarboxymethylcellulose (“belly jelly”) to the abdomen (FIGS. 12A-12E).

TABLE 1 Surgical Approaches of Applying Patch Grafting or InjectionGrafting onto Pig Livers Direct injection under the Details for Patchgraft directly on Direct injection into liver Periductal injection atcapsule around the bite treatment liver surface parenchyma ductalbifurcation ducts (e.g. common bite duct) Animal in group N = 28 N = 3 N= 3 N = 3 Cells per grafts* −5 × 10⁷ to 10⁸ 0.5-1 × 10⁷ per ml 0.5-2 ×10⁷ per 0.5 ml 0.5-2 × 10⁷ per 0.5 ml approaches by multiple infectionsOutcomes Good Good Good, but limited due to Bad (MELD Score**) effectsof swelling of the hydrogel Limitations/ Safe for normal animals Cellnumbers (dose) per Cell numbers (dose) per Restated to occlusion ofducts contraindications and for ones with liver injection is limited***,injection is limited due to and subsequent cholestasis injuroesl;efficient for multiple injections might limitation of proper symptoms.delivery of a large number address the problem. injection sites at whichof donor cells; the However, these would swelling will not causecomponents of patch cause higher risk for problems. Multiple grafts canbe adapted as bleeding. injections do not needed for compensate.Indications for Recommended for all Potential to be used for Recommendedfor left Not recommended Application stages of liver inborn errors oflobe grafts dysfunctions metabolism and early stage cirrhosis *Cells pergrafts in the study is showed as cell numbers per ml cross-linked HA**MELD Score (Model For End-Stage Liver Disease): Creatinine Bilirubin,INR, Sodium ***In this study, for 10 kg BW healthy piglet recipient, nomore than 0.5 ml per injection was recommended.

Two forms of SERI Silk (Sofregen, Medford, Mass.) provided the bestcombination of mechanical support and minimal adhesions (Table 2), aneffect further enhanced by application of the 2×HA to the outside (thefree side) surface of the silk backing after attachment to the targetsite. The product is a purified fibroin of Bombyx moth silk and knittedinto a scaffold to provide soft-tissue support. The stiffness of theoriginal version of Seri-Silk made it difficult to apply to sites withsignificant curvature. In later studies, we made use of “Contour SeriSilk” (Sofregen, Medford, Mass.) with considerably more flexibilityenabling the application of grafts to target sites with any degree ofcurvature. In grafts at 3 weeks, SERI-Silk was enveloped by collagenbands suggesting a mild fibrosis.

TABLE 2 Comparison of Backings tested for Patch Grafts. Ease in AdverseBackings handling reactions Adhesions** Information on backing SERI ®Surgical Scaffold Silk and Yes No Minimal Bombyx Moth Silk ContourSurgical Silk Scaffolds David Kaplan (Tuft's University, Boston, MA),Filamentous Silk Mesh Yes Yes Severe Sofregen (Boston, MA) Vetrix BioSISECM Yes Tended to 2-3 Regenerative Medicine Tech. Surgisis ®ES ™ SoftTissue Graft Yes become 2-3 Discontinued or renamed Alloderm Yesdislodged and 2-3 Decellularized tissue from human dermis Vicryl KnittedMesh Yes to fold over, Severe Ethicon Necrosis, Discoloration, &Fibrosis Seprafilm Too No 2 https://www.seprafilm.us/ fragile ReglydeToo No 2 http://www.biotimeinc.com/ fragile **Adhesions: These ranged toextent of severity. We assigned a number to indicate that severity; 0 =no adhesions; 1 = thin and easily disrupted adhesions; 2 = adhesionsrequiring blunt force dissection to disrupt; 3 = Dense adhesions thatwere dispersed only with the use of considerable force, resulting inpartial or total injury to the viscera. The most severe adhesionsobserved with any of the backings were those when used in combinationwith “belly jelly”, carboxymethylcellulose. Coating of serosal (outside)surface of backing with more concentrated (2X) HA reduced and minimizedadhesions Immunosuppression: All pigs received oral dosages of theimmunosuppressive drugs Tacrolimus (0.5 mg/kg) and Mycophenolate (500mg) twice daily, beginning 24 hours prior to surgery, and continuouslygiven thorough the post-surgical period.

Evidence for engraftment at one week after surgery was validated withTrichrome staining (FIGS. 3A and 3B) and with hematoxylin/eosin (H&E)staining (FIGS. 3A2 and 3B2) and demonstrated a remodeling of theGlisson Capsule plus a surprisingly broad region of remodeling of theparenchymal tissue beneath the site of the graft (see also FIGS. 9A-9B).In this region, the histological structures were lost entirely or werein the process of dissolution (see also FIGS. 6E-6H). Reconstitution ofthe Glisson capsule and of the lobular architecture occurred by 3 weeksfollowing resorption of HAs that led to maturation of the donor cellsand muting of the MMPs expression (FIG. 3B). Engraftment into the liversof NRG/FAH mice was completed and with cells uniformly throughout thetissue by 3-4 weeks post-surgery (FIG. 3C, and FIGS. 5A-5P).

In some embodiments, donor organoids of BTSCs/ELSMCs deriving fromtransgenic GFP+ pigs were grafted into wild type pigs (FIGS. 3A and 3B,4A) or into NRG/FAH mice (FIGS. 3C, 5L-5P) and were identified by GFPexpression (FIGS. 3C, and 8A). In liver, autofluorescence may derivefrom many different molecules (e.g. aromatic amino acids, flavins,vitamin A, lipofuscins). The autofluorescence for lipofuscins peak atwavelengths overlapping with that for GFP (FIG. 8A). Therefore,identification of donor cells in livers may be performed with anantibody to GFP (Rabbit anti-GFP antibody; Novus, NB600-308) and coupledto a secondary antibody with a red fluoroprobe (Donkey anti-rabbit 555,Invitrogen) causing donor cells to have a pink nucleus (FIGS. 4A and10), a merge of the red fluoroprobe with the blue from DAPI) in thediploid cells and a punctate pink entity associated with the blue nucleiin cells with larger nuclei (probable polyploid cells). In someembodiments, host cells were recognized given their blue nuclei butwithout GFP expression (FIGS. 3C, 4A, 10). Large numbers of donor GFP+BTSCS/ELSMCs were observed in the host liver near to the graft (FIG.4A). In some embodiments, donor GFP+ BTSCs/ELSMCs were observed alsonear host lobules on the cross-lobe side (FIGS. 4B and 10) that wasapproximately 1.5 cm from the graft site. Thus, the cells were in someembodiments able to migrate the entire width of the lobule in a week.

The liver lobules of mature hepatocytes contain lipofuscins in thecytosol with an autofluorescence green color. However, in young animals,the amount of lipofuscins is minimal and was easily distinguished fromthe nuclear GFP label (FIGS. 4B, 4Cii, and 8A). In some embodiments,Donor GFP+ cells that had matured were recognized as aggregates ofhepatocytes with pink nuclei (FIGS. 4C, 4Ci).

In some embodiments, upon transplantation of the patch grafts ofBTSCs/ELSMCs organoids resulted in remodeling of the Glisson Capsule andof tissue near to the graft site, followed by a merger of host and donorcells within a week (FIGS. 3A-3C-5A-5P, 6E-6H). Hematoxylin/Eosin (H&E)staining of the region of remodeling implicated inflammatory responsesinvolving both donor and host cells (FIGS. 6E-6H, 9A-9B). The GFP labelwas found only in the nuclei of the GFP+ BGTSCs/ELSMCs in the graft andin the nuclei of the GFP+ adult cells (FIGS. 4A-4E); however, there wasalso some cytoplasmic staining of GFP at week one in maturing cells,transitioning into entirely or primarily nuclear within another week ortwo (FIGS. 4A-4E, 5A-5P).

In some embodiments, integration of the donor cells within large regionsof the liver was completed by 2 weeks by which time HAs had been mostlyresorbed, and some donor cells had lineage restricted into adult hepaticcell fates comprising cholangiocytes (pan cytokeratin, pCK) andhepatocytes (albumin) (FIGS. 4A-4E). In some embodiments, donor GFP+cells were present throughout the organ and had acquired classicsinusoidal plates or ductular morphologies (FIG. 4E) and expressedintermediate (e.g. SOX9, alpha-fetoprotein, HNF4a) and adult (albumin,pCK) functions (FIG. 4E).

Engraftment efficiency by patch grafting resulted in essentially all ofthe donor cells transplanting into the host liver and remaining viable.They were not found in the remnants of the grafts at the liver surfaceof the liver, and there was no evidence of ectopic cell distribution toother organs (e.g. lung). The speed of migration of donor cells in theBTSC/ELSMC grafts through the liver resulted in donor cells in mostregions of the organ (liver) by the end of a week and with uniformlydispersed cells throughout the tissue (liver) by 2-3 weeks (FIGS.3A-3C-5A-5P).

In some embodiments, the present disclosure provides methods of rescuinghosts from a disease state by cellular engraftment. In one embodiment ofthe present disclosure, the method of rescuing a murine model of type Ityrosinemia due to deficiency in furmaryl-acetoacetate-hydrolase(NRG/FAH) mice were rescued by cell engraftment. NRG/FAH were obtainedfrom Dr. Lishan Su (Department of Microbiology and Immunology at UNC).NRG/FAH mice were established using CRISPR/Cas9 technology inimmunocompromised mice to achieve a murine model of type I tyrosinemiadue to deficiency in furmaryl-acetoacetate-hydrolase (FAH) and, inparallel, permissive for xenografts. The mice were maintained under theroutine conditions for immunocompromised hosts and were sustained withnormal livers and normal kidneys by being supplied a drug,2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC),Nitisinone, in their water (20 μg/ml). Nitisinone has been shown toblock the tyrosine pathway prior to the FAH enzymatic deficiency and soprevent the buildup of toxic intermediates affecting both the liver andthe kidney. NRG/FAH mice were treated with patch grafts of organoids ofporcine BTSC/ELSMCs and then weaned from Nitisinone. Controls were givenpatch grafts without cells and also weaned from Nitisinone.

All the animals with patch grafts of porcine BTSC/ELSMCs remainedhealthy for 4 weeks or more and gained weight (˜1 gm/every 2-3 days)after withdrawal of Nitisinone, whereas the controls (given patch graftswithout cells and weaned from Nitisinone) began losing weight (˜1gm/day) by day 17 post grafting (FIG. 11). The control mice with patchgrafts with no cells had to be euthanized by day 17; those with patchgrafts containing BTSCs/ELSMCs were euthanized on day 30. Histologicalassays indicated that mice with patch grafts with BTSCs/ELSMCs havelivers and kidneys similar to those of animals maintained on Nitisinone(FIGS. 5A, 5B, 5N). By contrast, there were massive injuries in thelivers and kidneys of animals with control grafts with no cells (FIGS.5C, 5D, 5N). The effects on livers and kidneys are known due to toxinsfrom tyrosine metabolism in hosts with FAH deficiency. The histologicalassays showed no cells or very limited number of cells remaining in thepatch grafts (FIG. 5E). GFP signals from porcine donor cells wereobserved throughout the grafted liver (FIGS. 3C, 5G, 5H, 5I) and thoughthere was some cytoplasmic staining of GFP, most was expressed in thenucleus; moreover, independent assays for H2B histone showed that itco-expressed with GFP whether in the nucleus or the cytoplasm (FIG. 5P).In addition to normal liver and kidney histology in animals with patchgrafts with BTSCs/ELSMCs, donor cells within the host livers were shownto express porcine FAH (FIG. 5O).

In some embodiments, the patch graft of the present disclosure maintainsstem cells in an immature state that maintains expression ofmatrix-metal-proteinases (MMPs) in the donor stem cells. The remodelingof the Glisson capsule and of the neighboring liver lobules correlatedwith elevated expression of multiple MMPs, enzymes known to dissolveextracellular matrix components and to be associated with cellmigration. FIGS. 6A-6H summarizes data from RNA-seq studies and IHCassays on MMPs expressed by stem/progenitors versus adult cells. BTSCsexpressed high levels of multiple MMPs, comprised of both secreted forms(e.g. MMP2, MMP7) as well as membrane-associated forms (e.g. MMP14,MMP15). The ELSMCs, precursors of endothelia and of stellate cells, alsocontributed to expression of multiple MMPs.

The findings from RNA-seq data were confirmed by IHC assays for theproteins (enzymes) encoded by MMP genes (FIGS. 6E-6H). IHC assaysconfirmed the presence of the secreted forms of MMPs such as MMP1, MMP2,MMP7, and MMP9, especially in the regions of remodeling. Proteinexpression of MMP1 was found in BTSCs/ELSMCs organoids and also inremodeling regions of grafts. However, existing data banks of RNA-seqfindings do not include MMP1 because of a lack of an annotated speciesof porcine MMP1 to be used for the analyses. Therefore, recognition ofits presence in the remodeling zones is based on the IHC assays.

Factors causing differentiation of donor cells resulted in muting ofexpression of secreted MMPs and, in parallel, a loss in potential forengraftment and migration (data not shown). These included serum,various soluble regulatory signals (e.g. growth factors, cytokines,hormones) known to influence differentiation of the donor cells,extracellular matrix components whether in the hydrogels or in thebackings (especially type I collagen), and the stiffness of the HAhydrogels (e.g. Pascal levels above ˜0.200-300). If differentiation ofELSMCs progressed preferentially to stroma (e.g. in the presence ofserum), the grafts became fibrotic; if to endothelia (in the presence offactors promoting angiogenesis), the grafts contained viable cells butremained superficial to the organ capsule (data not shown)

The present disclosure provided a novel method for transplantation ofcells into solid organs or tissues, including internal organs, anddemonstrated here with studies on liver. The method is safe, avoidsemboli and ectopic cell distribution, and optimizes rapid engraftmentand distribution of large numbers of cells throughout the host tissue.Within a week, there was engraftment of all of the donor cells(≥10^(8th) in pigs; ≥10^(6th) in mice) throughout significant portionsof the liver followed by migration and integration of cells atsignificant distances from graft sites. Maturation to mature cell typesoccurred throughout regions of engrafted cells by 2 weeks, followed byrestoration of Glisson's capsules and of tissue histology andarchitecture by 3 weeks. Importantly, patch grafts were able to rescueanimals from a disease state, demonstrated here with patch grafts inmice with type I tyrosinemia.

The presently disclosed methods are superior to methods of transplantingcells into solid organs by direct injection or by delivering cells via avascular route. These past methods of transplantation resulted in smallnumbers of cells being engrafted, in risks of emboli that can be lifethreatening, and in significant levels of ectopic cell distribution ofprobable but unknown significance clinically. These problems have causedcell therapies for internal solid organs to be used minimally or not atall

The present disclosure found that organoids provide the most successfularrangement for the cells for grafting to ensure appropriate,lineage-stage-specific epithelial-mesenchymal partnering. Accordingly,in some preferable embodiments, the donor cell mixture is formed byletting them self-select into organoids, after removal by panning ofmature mesenchymal. In some embodiments, the donor cell mixture may beformed by co-transplanting epithelial-mesenchymal cell partners by flowcytometrically immuno-selecting the relevant cells using theirdistinctive surface antigens from cell suspensions and then mixing theBTSCs and the ELSMCs according to the ratios found in cells suspensionsfrom freshly isolated tissues. Without being bound by theory, it is ahypothesis of the present disclosure that establishinglineage-stage-appropriate epithelial-mesenchymal partners with providesrelevant paracrine signaling for the grafts, and yields organoids underdefined (serum-free) conditions, that made them easily cultured orcryopreserved.

In one aspect, the primary design of the grafts consisted of mixing ofcells with appropriate biomaterials that can form a hydrogel that keepscells localized to the target site. The cells in the soft hydrogels wereprotected with a backing that is neutral regarding effects on the donorcells. A preferable embodiment of the graft biomaterials may benon-sulfated or minimally sulfated glycosaminoglycans (GAGs), such ashyaluronans (HAs), found in all stem cell niches, with receptors to HAsbeing classic stem cell traits. Without being bound by theory, it is ahypothesis of the present disclosure that HAs supported maintenance ofcells as immature (i.e. as stem/progenitors), and optimized theirexpression of secreted MMPs essential for engraftment and for migrationand integration into the host tissue.

In some embodiments, the present disclosure demonstrated that thedisclosed methods induced engraftment processes resulting in remodelingof the Glisson Capsule and of the host tissue near to the graft (FIGS.3A-3C, 5A-5P, and 9A-9B). To validate the findings of remodeling,Trichrome staining was used and its staining of extracellular matrixcomponents (FIG. 3A-3C) plus staining with H&E (FIGS. 3A-3C and FIGS.9A-9B) confirmed remodeling phenomena associated with inflammation. By 3weeks post-surgery and following clearance of HAs, there wasreconstitution of the Glisson's capsules and of the normal tissuehistology. The remodeling zone (see FIGS. 3A-3C-5A-5P) was shown toinvolve multiple forms of MMPs (FIGS. 6A-H) and to be transientreverting to normal histology by 3 weeks.

Although there are multiple types of HAs, thiol-modified ones can betriggered with PEGDA to crosslink to form a hydrogel with precisebiochemical and mechanical properties. These HA hydrogels are clinicallyuseful for cell and molecule delivery in vivo. The properties of HAs arereproducible, stable, confer elasticity, allow access into the graft ofall soluble signals in blood, lymph or interstitial fluid, and minimizethe maturation of donor cells until engraftment and migration haveoccurred. The ability to vary the rheological factors with simplechanges in HA and PEGDA concentrations enables “tuning” of the HAproperties, and provides additional advantages in guiding the directionof migration of the cells and in minimizing adhesions. Soft HAhydrogels, ones mimicking properties in stem cell niches were permissivefor expression of the stem/progenitor cells-associated repertoire ofMMPs, especially the secreted MMPs. Thus, mechanical properties of HAs,studied for years in functions of skeletal tissues are important also inmanaging grafting strategies.

Patch grafts containing stem/progenitors resulted in striking phenomenaof grafts “melting” into tissues within a few days, followed by a mergerof donor and host cells, and a distribution of cells throughoutsignificant regions of the organ within a week (see FIGS. 3A-3C-5A-5P).During the remodeling phase, primarily the first weekpost-transplantation, there was often cytoplasmic expression of the GFPlabel in the donor cells, especially if they had lineage restricted toan adult fate (FIG. 4C). This was concerning, since the GFP label istagged to the H-2B histone locus meaning that it should have been foundonly in the nucleus. Without being bound by theory, it is an hypothesisof the present disclosure that histones can be found in the cytoplasmduring inflammatory processes, and that there was abundant evidence forsuch inflammatory processes in the remodeling zones (FIGS. 9A-9B), butthese inflammatory processes diminished with time post-transplantationresulting in reconstitution of the Glisson Capsule, stabilizedhistological structures, and with donor cells having primarily orentirely nuclear GFP staining by 3-4 weeks (FIGS. 4D, 4E, 5G). Incontrol studies, antibody to histone yielded similar patterns as the onefor GFP (FIG. 5P).

It is a discovery of the present disclosure that the engraftment andintegration process correlated with expression of multiple MMPs, afamily of calcium-dependent, zinc-containing endopeptidases that degradeextracellular matrix components.

Immature cells express express high levels of secreted forms (e.g. MMP2,MMP7) as well as membrane-associated forms (e.g. MMP14, MMP15). IHCassays indicated that protein levels of secreted MMPs (e.g. MMP1, MMP2,MMP7) were found richly expressed in areas of remodeling (FIGS. 6A-6H).

The biomaterials of the grafts, especially the HAs, have been shown exvivo and in vivo to maintain sternness traits in cells. Since the graftsare devoid of known signals that can trigger fate determination, thefindings of donor cells that had matured into distinct adult fatesimplicate the local microenvironment of the host tissue as the logicalsource of relevant factors for dictating fates in the maturationalprocesses.

Patch graft strategies are safe and effective for transplantation oflarge numbers of cells into a solid organ or tissue and so offerreplacement of missing functions or alleviation of disease states. Thenumbers of cells per patch that can be engrafted are considerable (10⁸in pigs; 10⁷ in mice) and dictated by the dimensions of the patch graftand the numbers of organoids. These findings are in contrast to thelimited numbers of cells (e.g. ≤10⁶) feasible with vascular delivery orby injection grafting.

Conditions (soluble growth factors, cytokines, serum, matrix components,mechanical forces) that caused donor cells to differentiate resulted inreduction in secreted MMPs and, in parallel, abrogation of theengraftment and migration process. Complementing these findings werecontrol studies with patch grafts of mature hepatocytes partnered withendothelia; the donor cells survived and were functional, but they didnot engraft (data not shown). Therefore, engraftment requires a sourceof secreted MMPs, ideally a cellular source that can interactdynamically to generate the multiple forms of secreted as well as plasmamembrane-associated MMPs. The mature cells are unable to do this, sincethey expressed only or primarily the plasma membrane-associated forms(FIGS. 6A-6H).

This approach offers alternative methods for cell therapies. It provedsafe as long as biomaterials and the backing used were neutral withrespect to the host tissue and collectively supportive of maintenance ofthe donor cells as immature and so able to produce the relevantrepertoire of MMPs required for engraftment, migration, and integration.

Abbreviations

ADHEP, adult hepatocytes; AFP, α-fetoprotein; ALB, albumin; BTSCs,biliary tree stem cells; CD, common determinant; CD44, hyaluronanreceptors; CD133, prominin; Cdx2, caudal type homeobox 2; CFTR, cysticfibrosis transmembrane conductance regulator; CK, cytokeratin protein;CXCR4, CXC-chemokine receptor 4 (also called fusin or CD184; also calledplatelet factor 4; EGF, epidermal growth factor; ELSMCs, early lineagestage mesenchymal cells, consisting of angioblasts and theirdescendants, precursors to endothelia and to stellate cells; EpCAM,epithelial cell adhesion molecule; FAH, fumaryl-acetoacetate hydrolase,an enzyme critical to tyrosine metabolism (its absence results in type Ityrosinemia); FGF, fibroblast growth factor; HBs, hepatoblasts; HGF,hepatocyte growth factor; HpSCs, hepatic stem cells; KM, Kubota'sMedium, a serum-free medium designed for endodermal stem cells; KRT,cytokeratin gene; LGR5, Leucine-rich repeat-containing G-protein coupledreceptor 5 that binds to R-spondin; Mafa, V-Maf MusculoaponeuroticFibrosarcoma Oncogene Homolog A; MMPs, matrix metallo-proteinases, alarge family of proteinases associated with dissolution of extracellularmatrix, with cell migration and with regenerative responses; NANOG, atranscription factor critically involved with self-renewal; NCAM, neuralcell adhesion molecule; NGN, neurogenin; NRG,NOD-Rag1−/−IL2RgammaC-null; NIS, sodium/iodide symporter; OCT4,(octamer-binding transcription factor 4) also known as POU5F1 (POUdomain, class 5, transcription factor 1), a gene expressed by stemcells; PDX1, pancreatic and duodenal homeobox 1, a transcription factorcritical for pancreatic development; PBGs, peribiliary glands, stem cellniches for biliary tree stem cells; SALL4, Sal-like protein 4 found tobe important for self-replication of stem cells; SOX, Sry-related HMGbox; SOX2, a transcription factor that is essential for maintainingself-renewal, or pluripotency in embryonic and determined stem cells.SOX9, transcription factor associated with endodermal tissues (liver,gut and pancreas; SOX17, a transcription factor essential fordifferentiation of liver; VEGF, vascular endothelial cell growth factor.

WORKING EXAMPLES Example 1: Preparation and Characterization of PatchGrafts

This example describes an exemplary method for preparing andcharacterizing patch grafts.

Materials

Companies providing equipment, reagents and/or supplies: Abcam,Cambridge, Mass.; ACD Labs, Toronto, CA; Acris Antibodies, Inc), SanDiego, Calif.; Advanced Bioscience Resources Inc) (ABR), Rockville, Md.;Agilent Technologies, Santa Clara, Calif.; Alpco Diagnostics, Salem,N.H.; Applied Biosystems, Foster City, Calif.; BD Pharmingen, San Jose,Calif.; Becton Dickenson, Franklin Lakes, N.J.; Bethyl Laboratories,Montgomery, Tex.; BioAssay Systems, Hayward, Calif.; Cambridge IsotopeLaboratories, Tewksbury, Mass.; Biotime, Alameda, Calif.; Carl ZeissMicroscopy, Thornwood, N.Y.; Carolina Liquid Chemistries, Corp.,Winston-Salem, N.C.; Charles River Laboratories International, Inc),Wilmington, Mass.; Chenomx, Alberta, Canada; Cole-Parmer, Court VernonHills, Ill.; DiaPharma, West Chester Township, Ohio; Fisher Scientific,Pittsburgh, Pa.; Gatan, Inc), Pleasanton, Calif.; Illumina, San Diego,Calif.; Ingenuity, Redwood City, Calif.; Life Technologies Corp., GrandIsland, N.Y.; Leica, Washington, D.C.; LifeSpan Biosciences, Inc),Seattle, A; Molecular Devices, Sunnyvale, Calif.; Olympus ScientificSolutions Americas Corp., Waltham, Mass.; PhoenixSongs Biologicals(PSB), Branford, Conn.; Polysciences, Inc), Warrington, Pa.; Qiagen,Germantown, Md.; R&D Systems, Minneapolis, Minn.; RayBiotech, Norcross,Ga.; Roche Diagnostics, Mannheim, Germany; Santa Cruz Biotechnology,Inc), Dallas, Tex.; Sigma-Aldrich, St. Louis, Mo.; Sofregen, Medford,Mass.; Takara, Otsu, Japan; Tousimis Research Corp., Rockville, Md.;Triangle Research Labs (TRL), Research Triangle Park, N.C.; Umetrics,Umea, Sweden; Varian Medical Systems, Inc), Palo Alto, Calif.; VectorLaboratories, Burlingame, Calif.; VWR Scientific, Radnor, Pa.)

Animals

Location of Facilities.

Animals used as hosts or as donors for cells were maintained infacilities at the College of Veterinary Medicine at NCSU (Raleigh,N.C.). Surgeries, necropsies, and the collection of all biologicalfluids and tissues were performed at these facilities. All procedureswere approved by the IACUC committee at NCSU.

Pig Hosts Used for the Grafts.

The pigs being used as recipients were a mixture of six differentbreeds: a six-way cross consisting of Yorkshires, Large Whites,Landraces (from the sows), Durocs, Spots, and Pietrans (from the boars).This highly heterogeneous genetic background is desirable in that itparallels the heterogeneous genetic constitutions of human populations.The host animals were all females, approximately six weeks of age and˜15 kg.

Pig Donors for Cells.

There were two categories: a) transgenic donor animals carrying a GFPtransgene (all of the studies in this report) and b) male pigs,approximately six weeks of age and ˜15 kg, were used as donors for celltransplantation into females (parallel studies with findings to reportedin subsequent reports). The GFP+ donor animals were obtained by breedinga transgenic H2B-GFP boar with a wild type gilt by standard artificialinsemination. The model was developed via CRISPR-Cas9 mediatedhomology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenousβ-actin (ACTB) locus. The transgenic animals show ubiquitous expressionof pH2B-eGFP in all tissues.

Fusion of the GFP to H2B results in localization of the GFP marker tothe nucleosome and allows clear nuclear visualization as well as thestudy of chromosome dynamics. The founder line has been analyzedextensively and ubiquitous and nuclear localized expression has beenconfirmed. In addition, breeding has demonstrated transmission of theH2B-GFP to the next generation. All animals were healthy, and multiplepregnancies have been established with progeny showing the expectedMendelian ratio for the transmission of the pH2B-eGFP. The maleoffspring were genotyped at birth, and those that were positive for thetransgene were humanely euthanized for tissue collection, and isolationof donor cells.

Genotyping of Animals.

For each donor and recipient animal, the swine leucocyte antigen class I(SLA-I) and class II (SLA-II) loci have been PCR amplified using primersdesigned to amplify known alleles in these regions based on thePCR-sequence-specific-primer strategy. The system consists of 47discriminatory SLA-I primer sets amplifying the SLA-1, SLA-2, and SLA-3loci63, and 47 discriminatory SLA-II primer sets amplifying the DRB 1,DQB1, and DQA loci. These primer sets have been developed todifferentiate alleles by groups that share similar sequence motifs, andhave been shown easily and unambiguously to detect known SLA-I andSLA-II alleles. When used together, these primer sets effectivelyprovided a haplotype for each animal that was tested, thus providing anassay to confirm easily a matched or mismatched haplotype in donor andrecipient animals.

Breeding pairs of NRG/FAH Mice were obtained from Dr. Lishan Su(Department of Microbiology and Immunology, UNC, Chapel Hill, N.C.) andmaintained in the animal facilities at UNC (Chapel Hill, N.C.) underconditions appropriate for immunocompromised hosts. These FAH mice wereestablished by CRISPR/Cas 9 technology using NRG mice, meaning that themice are immunocompromised and secondarily have been made deficient infumaryl acetoacetate hydrolase (FAH). The FAH gene encodes fumarylacetoacetate hydrolase that is the final enzyme in the tyrosine andphenylalanine catabolism pathway. FAH is expressed highly in liver andkidney cells and less so in endocrine tissues. NRG (NOD-Rag1−/−IL2RgammaC-null)/FAH (fumarylacetoacetate hydrolase) knockout mice showa progressive liver (and kidney) damage phenotype that mimics majorfeatures of hereditary tyrosinemia type 1 (HT1) in humans, includingtyrosinemia, appearance of succinylacetone in blood and urine, and liverand kidney injuries The FAH deficiency complications in mice weremanaged by adding (20 μg/ml) of2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; alsocalled Nitisinone) to the water supply for the animals. NTBC suspendsproduction of toxic metabolites in tyrosine catabolism pathway which iscaused by the absence of FAH. The livers and kidneys of these micedemonstrate normal histology unless the animals are provided normalwater without Nitisinone. When presented with regular water, evidence ofsevere tyrosinemia occurs within 2 weeks and results in a need toeuthanize the mice by 3 weeks.

Media and Solutions

All media were sterile-filtered (0.22 μm filter) and kept in the dark at4° C. before use. Basal medium and fetal bovine serum (FBS) werepurchased from GIBCO/Invitrogen. All growth factors were purchased fromR&D Systems. All other reagents, except those noted, were obtained fromSigma.

Cell Wash.

599 mls of basal medium (e.g. RPMI 1640; Gibco #11875-093) wassupplemented with 0.5 grams of serum albumin (Sigma, # A8896-5G,fatty-acid-free), 10-9 M selenium, and 5 mis of antibiotics (Gibco#35240-062, AAS). It was used for washing tissues and cells duringprocessing.

Collagenase Buffer.

Consists of 100 mls of cell wash supplemented with collagenase (Sigma #C5138) with a final concentration of 600 U/ml (R1451 25 mg) for biliarytree (ducts) tissue and 300 Um′ (12.5 mg) for organs (e.g. liver)

Kubota's Medium, a wholly defined, serum-free medium designed forendodermal stem/progenitors was used to prepare cell suspensions,organoids and HA hydrogels.

This medium consists of any basal medium (here being RPMI 1640) with nocopper, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin(purified, fatty-acid-free; fraction V), 4.5 mM nicotinamide, 0.1 nMzinc sulfate heptahydrate, 5 μg/ml transferrin/Fe, 5 μg/ml insulin, 10μg/ml high density lipoprotein, and a mixture of purified free fattyacids that are presented complexed with fatty acid-free, highly purifiedalbumin. Its preparation is given in detail in a methods review67. Also,it is available commercially from PhoenixSongs Biologicals (Branford,Conn.).

Hyaluronans (HAs).

Soluble, long chain forms of HA (Sigma Catalog #52747) were used instabilization of organoid cultures and in cryopreservation. Those usedto make the hydrogels, thiol-modified HAs, were obtained from GlycosanBiosciences, a subsidiary of Biotime. The components for thesethiol-modified HAs were made by a proprietary bacterial-fermentationprocess using Bacillus subtilis as the host in an ISO 9001:2000 process(www.biopolymer.novozymes.com/).

The components were produced by Novozymes under the trade name HyaCare®and are 100% free of animal-derived materials and residual organicsolvent. No animal-derived ingredients are used in the production, andthere are very low protein levels and no endotoxins. The productionfollows the standards set by the European Pharmacopoeia). The HAhydrogels were prepared using Glycosil (HyStem® HAs, ESI BIO-CG313), thethiol-modified HAs, that can be trigged to form disulfide bridges in thepresence of oxygen, or by forming thio-ether linkages using polyethyleneglycol diacrylate (PEGDA). Glycosil® is reconstituted as a 1% solutionof thiolated HA in 1% phosphate buffered saline (PBS) using degassedwater, or, in our case, in serum-free Kubota's Medium. Uponreconstitution, it remains liquid for several hours but can undergo somegelation if exposed to oxygen. More precise gelation occurs with notemperature or pH changes if Glycosil is treated with a cross-linkersuch as PEGDA causing gelation to occur within a couple of minutes.

The level of cross-linking is the main contributor to the level ofstiffness, or rigidity, and can be controlled by adjusting the ratio ofthe thiol-modified HAs to PEGDA. In prior studies, stem cell populationswere tested in HA hydrogels of varying level of rigidity and were foundto remain as stem cells, both antigenically and functionally (e.g., withrespect to ability to migrate), only if the level of rigidity was lessthan 100-200 Pa We made use of this finding to design the grafts with avery soft layer and with more rigid layers of hyaluronan hydrogels inthe backing to form a barrier to migration in directions other than thetarget tissue as well as to minimize adhesions from cells from nearbytissues. The 3 versions of the hydrogels with distinct levels ofrigidity are characterized in FIGS. 2A-2E, characterizations thatincluded direct measurements of the rheological properties. The mostrigid barrier, that of the 10×HA hydrogel (rigidity=760 Pa), wasprepared on the backing ahead of time and could be cryopreserved ifdesired. At the time of the surgery, the donor cells were prepared inthe soft, 1×HA hydrogel (rigidity=60 Pa); placed onto the more rigid 10×hydrogel (already on the backing); and the patch tethered to the targetsite. After tethering, the outside of the graft was coated or paintedwith the 2X HA hydrogel (rigidity=106 Pa) using a NORM-JECT 4010.200V0Plastic Syringe with a BD Micro-Fine™ IV permanently attached needle.

Macro-scale rheological properties of hydrogels were determined using astress-controlled cone-and-plate rheometer (TA Instruments, AR-G2, 40 mmcone diameter, 1° angle). Gels actively polymerized on the rheometerwhile oscillating at 1 rad/s frequency and 0.6 Pa stress amplitude withthe modulus monitored continuously to query for sufficient completion ofthe cross-linking reaction.

Once equilibrated, the hydrogels were subjected to an oscillatoryfrequency sweep (stress amplitude: 0.6 Pa, frequency range: 0.01-100Hz). The rheological properties of the 3 versions of hyaluronanhydrogels that were used are summarized in FIG. 2A-2E and comprised softhydrogels (˜100 Pa), more rigid ones (˜700 Pa) and intermediate levels(˜200-300 Pa).

Donor Cells

Donor cells were derived from transgenic H2B-GFP pigs as describedabove. They offer a significant advantage for cell transplantationstudies in that all cells are tagged with H2B-GFP fusion protein. Theuse of fluorescent proteins as molecular tags enabled the donor cells tobe tracked in their migration and engraftment after transplantation.This fusion protein is targeted to the nucleosomes by fusing GFP withthe nucleosomal H2B protein, resulting in a nuclear/chromatin GFPsignal.

In the characterizations of the grafts, autofluorescence both of thesilk backing (spring green color) and also of lipofuscins (forest darkgreen color) in mature hepatocytes presented a challenge given theoverlap in wavelengths with those of GFP. Therefore, we shifted the GFP+signal to a pink or rose color using an antibody to GFP and secondarilyto an antibody with a red fluoroprobe (FIGS. 4A-4E, 10). This resultedin the stem cells being recognized as small cells with pink nuclei(merger of the nuclear blue DAPI staining with the antibody-tagged-rosecolored GFP+ label). Any donor cells that matured into hepatocytes wererecognized as having pink nuclei and with lipofuscin autofluorescence inthe cytoplasm (FIGS. 4A-4E). In the patch grafts onto mouse livers, somecells had nuclei that were entirely pink, and some had large blue nucleito which was associated a punctate pink entity (FIGS. 5A-5P). Wehypothesize that this is due to a mixture of donor cells (diploid) witha single pink nucleus and some donor cells that were maturing intopolyploid cells with larger nuclei and so may have had the GFP labellocalized to one region of the nucleus. Alternatively, those withdiscrete localization of the GFP label may represent a fusion of donorand host cells with young murine hepatocytes well known to have levelsof polyploidy ranging from 4N to 32N.

Preparation of Cells.

Porcine extrahepatic biliary tree tissue (gall bladder, common duct,hepatic ducts) were obtained from transgenic pigs. Tissues were poundedwith a sterilized, stainless steel mallet to eliminate the parenchymalcells, carefully keeping the linkage of the intra-hepatic andextrahepatic bile ducts. The biliary tree was then washed with the “cellwash” buffer comprised of a sterile, serum-free basal mediumsupplemented with antibiotics, 0.1% serum albumin, and 1 nM selenium(10-9M). It was then mechanically dissociated with crossed scalpels, andthe aggregates enzymatically dispersed into a cell suspension inRPMI-1640 supplemented with 0.1% bovine serum albumin (BSA), 1 nMselenium, 300 Um′ type IV collagenase, 0.3 mg/ml deoxyribonuclease(DNAse) and antibiotics. Digestion was done at 32° C. with frequentagitation for 30-60 minutes. Most tissues required two rounds ofdigestions followed by centrifugation at 1100 rpm at 4° C. Cell pelletswere combined and re-suspended in cell wash. The cell suspension wascentrifuged at 30 G for 5 minutes at 4° C. to remove red blood cells.The cell pellets were again re-suspended in cell wash and filteredthrough a 40 μm nylon cell strainer (Becton Dickenson Falcon #352340)and with fresh cell wash. The cell numbers were determined and viabilitywas assessed using Trypan Blue. Cell viability above 90-95% wasroutinely observed.

Mesenchymal Stem/Progenitors Needed as Partners.

In prior studies, we defined the antigenic profile of populations ofmesenchymal cells that provide critical paracrine signals needed forhepatic and biliary tree stem cells versus others required for matureparenchymal cells. The mesenchymal cells needed as partners for BTSCsare subpopulations devoid of MHC antigens, with low side scatter, andidentifiable as angioblasts (CD117+, CD133+, VEGF-receptor+, andnegative for CD31), precursors to endothelia (CD133+, VEGF-receptor+,and CD31+), and precursors to stellate cells (CD146+, ICAM1+, VCAM+,alpha-smooth muscle actin (ASMA)+, and negative for vitamin A). We referto these 3 subpopulations collectively as early lineage stagemesenchymal cells (ELSMCs).

By contrast, adult hepatocytes are associated with mature sinusoidalendothelia (CD31+++, type IV collagen+, VEGF-receptor+, and negative forCD117) and those for adult cholangiocytes that are associated withmature stellate and stromal cells (ICAM-1+, ASMA+, Vitamin A++, type Icollagen+).

Formation of Organoids.

The cell suspensions were added to Multiwell Flat Bottom Cell CulturePlates (Corning #353043) in serum-free Kubota's Medium and incubatedfor—an hour at 37° C. to facilitate attachment of mature mesenchymalcells. Mature mesenchymal cells attached to the dishes within 10-15minutes even though the medium was serum-free. The cells remaining insuspension were transferred to another dish and again incubated for upto an hour. Repeats of this resulted in depletion of a significantfraction of the mature mesenchymal cells. After depletion of maturemesenchymal cells, the remaining floating cells were seeded at ˜2×10⁵cells per wells in serum-free Kubota's Medium in Corning's ultralowattachment dishes (Corning #3471) and were incubated overnight at 37° C.in a CO₂ incubator. Organoids comprised of the biliary tree stem cells(BTSCs) and of ELMSCs formed overnight (FIGS. 1A-1D). These organoidcultures survived for weeks in Kubota's Medium, especially if the mediumwas supplemented (0.1%) with soluble forms of HAs (Sigma). They couldalso be cryopreserved as described below. From each gram of neonatal pigbiliary tree tissue, we obtained ˜1.5×10⁷ cells. We used ˜3-6×10⁵ cellsper well of a 6-well, ultra-low attachment plate and incubated in theserum-free Kubota's Medium. The cells produced, on average, 6000 to20,000 small organoids (˜50-100 cells/organoid/well). For the grafts, weused at least 100,000 organoids (>10⁷ cells). Depending on the size ofthe backing, we were able to increase the number of organoids in thegrafts up to 10⁸ organoids (i.e. ˜10⁹ cells) or more embedded in ˜1 mlof the soft hyaluronan hydrogel on a 3 cm×4.5 cm backing.

Cryopreservation of Stem Cell Organoids. Isolated stem cell organoidswere cryopreserved in Cryostor10, an isotonic cryopreservation buffercontaining antifreeze factors, dextran and DMSO (BioLife, Seattle,Wash.; https://www.stemcell.com/products/cryostor-cs10.html). Theviability of the cells was improved further with supplementation with0.1% HAs (Sigma #52747). Cryopreservation was done using CryoMed™Controlled-Rate Freezers. The viability on thawing was greater than 90%,and cells after thawing were able to form organoids or to attach, toexpand ex vivo and in vivo and to give rise to the expected mature cellsin vitro and in vivo.

Composition of the Grafts (FIGS. 1A-1D and 2A-2E). Isolating the cellsand assembling the grafts are characterized in a schematic in FIGS.1A-1D and FIG. 7 and with the details summarized in FIGS. 2A-2E. Thegrafts were formed by using a backing (Table 1, above) onto which wereplaced the stem cell organoids embedded in the soft hyaluronanhydrogels. These were readily prepared ahead of time and maintained in aculture dish in an incubator overnight. The grafts proved stable at thetarget site for the duration of the experiments. Cryopreservation of theorganoids was achieved readily, but that of the organoids when withinthe soft hydrogel was not. This meant that embedding the organoids inthe soft hydrogel had to be done just prior to surgery.

Surgery

Surgical Procedures.

Anesthesia was induced by administering a combination ofketamine/xylazine (2-3 mg/kg weight each) injected IV or 20 mg/kgketamine plus 2 gm/kg xylazine IM, and was maintained by isoflurane inoxygen administered via a closed-circuit gas anesthetic unit. Theanimals were positioned in dorsal recumbency, and the ventral abdomenwas clipped from xyphoid to pubis. The skin was aseptically preparedwith alternating iodinated scrub and alcohol solutions. After entry intothe surgery suite, preparation of the skin was repeated using steriletechnique, and the area was covered with a topical iodine solutionbefore application of sterile surgical drapes. The surgeons usedappropriate aseptic technique. A mid-ventral incision was made throughthe skin, through subcutaneous tissues and linea alba, starting at thexiphoid process and extending caudally 8-12 cm. The left hepaticdivision was exposed and a 3×4.5 cm patch graft was applied to theventral surface of the liver and containing 1×HA (˜60 Pa) with embeddedorganoids placed onto the backing containing 10×HA (˜760 Pa), and thepatch was placed in direct contact onto the surface of the livercapsule. The patch graft was sutured to the liver using 4-6 simple,interrupted sutures of 4-0 polypropylene. The exposed surface of thegraft was then treated with 2 mls of 2×HA hydrogel (˜106 Pa), a level ofrigidity that was fluid enough to permit it to be painted or coated ontothe outside of the graft; it served further to minimize adhesions fromneighboring tissues. Following placement of the surgical graft, thelinea alba was closed with a simple continuous suture using 0-PDS. Thelinea was blocked with 2 mg/kg 0.5% bupivacaine, IM. The subcutaneoustissues and skin were closed with continuous 2-0 PDS and 3-0 Monocrylsutures, respectively. Tissue adhesive was placed on the skin surface.

Immunosuppression.

The graft transplants from the transgenic pigs to the wild typerecipients were allogeneic and so required immunosuppression. Theimmune-suppression protocols used were ones established by others. Allpigs received oral dosages of the immunosuppressive drugs Tacrolimus(0.5 mg/kg) and Mycophenolate (500 mg) twice daily, beginning 24 hoursprior to surgery. The drugs were given continuously for the entireexperimental period. These could be given to the animals easily if mixedwith their favorite foods.

Necropsy Procedures.

All animals were humanely euthanized at the designated time point bysedation with Ketamine/Xylazine, and isofluorane anesthesia, followed byan intravenous injection of a lethal dose of sodium pentobarbital. Uponconfirmation of death, the carcass was carefully dissected, and thetarget organs were removed, and placed in chilled Kubota's Medium fortransportation to the lab. In addition to the liver, the lungs, heart,kidney, and spleen were collected and fixed in 10% neutral formalin.

Patch Grafts for NRG/FAH Mice.

Newborn piglet liver cells (106 H2B-GFP-cell mixture) were used to formorganoids that were embedded in 100 μl of 1×HA hydrogel, transferred to0.7 cm×1.2 cm Seri-silk contour backing impregnated with 10×HA hydrogelto form the patch graft. Patch grafts were then grafted onto the NRG/FAHmouse liver by sliding the graft between the medial and left laterallobe. Using a micropipette, surgical glue was used on the edge of thepatch to fasten the patch to the graft site. This was followed byapplying 200 μl of 2×HA hydrogel as an adhesion barrier.

The muscle layer and skin were closed using sutures or clips; 400 μlsaline were given to the mice for hydration; and 100 μl Buprenorphinewas given as post-surgical treatment. Controls received a patch withoutcells. All animals were subjected to surgery recovery overnight. On day1 post grafting, a 7-days-long-stepwise weaning process eliminating NTBCwas done (reductions to 25% on day 1; to 12% on day 2 to day 3; to 6%from day 4 to day 6, and to 0% on day 7). In later studies, we learnedthat this gradual weaning process is not required; one can simplyeliminate the drug within ˜24 hours after the surgical procedures forpatch grafting. Body weight measurements were used to check forabnormalities of the animals.

Characterizations of the Grafts

Histology.

After 48+ hours of fixation, tissues samples were placed in labeledcassettes in 70% ethanol and were processed on a long cycle at 60degrees in a Leica ASP300S Tissue Processor for approximately 10 hours.After completion of the overnight processing, samples were embeddedusing the Leica EG1160 Embedding Station. A mold was filled with wax andthe sample was placed in the correct orientation so that desiredsections could be collected. The cassette was chilled until the blockand tissue sample could be removed as one unit from the mold. The blockwas sectioned at 5 microns using a Leica RM2235 Microtome; the sectionswere floated in the water bath and placed onto slides. The slides wereallowed to air dry overnight before staining. Sections were stained forHaematoxylin and Eosin (H&E; Reagents #7211 and #7111) or Masson'sTrichrome (Masson's Trichrome Stain: Blue Collagen Kit#87019) usingRichard Allan Scientific Histology Products and following themanufacturer's recommended protocol; the protocol is programed into aLeica Autostainer XL.

Immunofluorescence (IF) of Unstained Frozen Sections of Liver. Issues ofAutofluorescence Sections of livers from wild type pigs and transgenicpigs (FIG. 8) were prepared from tissue that was embedded and frozen inOCT and flash frozen at ˜200 C for frozen sectioning. Frozen sectionswere imaged to observe the donor cells with GFP-linked to H2B (FIG. 8).The high autofluorescence in the cytoplasm of hepatocytes (lipofuscins)and the autofluorescence of the Seri-Silk backing caused problems invisualization of the GFP+ cells. In some embodiments, by preparingparaffin sections and staining for the GFP using a rabbit polyclonalantibody to GFP (Novus Biologicals, NE600-308); the rabbit anti-GFPantibody was used in combination with a secondary antibody of donkeyanti-rabbit IgG H&L (Alexa Fluor 568; ab175470, Invitrogen), whileDonkey anti-Goat IgG Alexa Fluor 488 antibody was used to excludenon-specific staining of hepatic autofluorescence (FIGS. 3C, 4, and 10).For immunofluorescence of GFP following treatment with an antibody toGFP, frozen sections were thawed for 1 hour at room temperature and thenfixed in 10% buffered formaldehyde, acetone or methanol. After fixation,sections were washed 3 times in 1% phosphate buffered saline (PBS),followed by blocking with 2.5% horse serum in PBS for 1 hour at roomtemperature. Primary antibodies diluted in 10% goat serum in PBS wereadded and incubated overnight at 4° C. The next morning, sections wererinsed 3 times with PBS and incubated with secondary antibodies dilutedin 2.5% horse serum in PBS for 2 hours at room temperature. Images weretaken using a Zeiss CLSM 710 Spectral Confocal Laser Scanning microscope(Carl Zeiss Microscopy). Antibodies are listed in Tables 3 and 4, below.Autofluorescence was reduced by quenching with the use of dyes and thatincluded Trypan Blue. Trypan Blue was used on tissues/cells at 0.4% inPBS. This reduces the background significantly.

For the images in FIGS. 4D and E (performed at Sapienza, Rome, Italy).Sections (3 μm) were stained with hematoxylin-eosin and Sirius red,according to standard protocols. For immunohistochemistry, endogenousperoxidase activity was blocked by a 30 min incubation in methanolichydrogen peroxide (2.5%). Antigens were retrieved, as indicated by thevendor, by applying Proteinase K (code S3020, Dako, Glostrup, Denmark)for 10 min at room temperature. Sections were then incubated overnightat 4° C. with primary antibodies (pan-Cytokeratin, Dako, code: Z0622,dilution: 1:100; Sox9, Millipore, code: AB5535, dilution: 1:200).

TABLE 3 Antibodies used for studies of organoids, in situ histologyassays, and for patch grafts Primary Antibodies Clonality, CatalogueDilution/ Category Antibody Host [clone #] Isotype Supplier NumberApplication Pluripotent EpCAM Rb Poly, IgG Abcam ab71916 IHC-P (1:200)and nonconjugated IF (1:200) Endodermal SOX9 Rb Poly, IgG ChemiconAB5535 ICC (1:800) transcript- nonconjugated IF (1:500) tion factor PDX1Gt Poly, IgG R&D AF2419 IHC-P/ICC (1:200) genes nonconjugated System IF(1:50) NIS Ms Mono, IgG1 Abcam ab17795 IHC-P/ICC (1:50) [SPM186] SOX17Ms Mono, IgG1 Abcam ab84990 IHC-P (1:100) [OTI3B10] IF/ICC (1:50) OCT4Gt Poly, IgG Santa SC-9081 IHC-P (1:100) nonconjugated CruzBiotechnology Pig liver Porcine Rb Poly, IgG Abcam ab79960 IHC-P (1:100)functional Albumin nonconjugated HNFA Ms Mono, IgG2a Abcam ab41898 IHC-P(1:200) [K9218] IF (1:800) GFP GFP Rb Poly, IgG Novus NB600-308 IHC-P(1:500) nonconjugated Biologicals IF (1:200) GFP-555 GFP Tag Rb Poly,IgG Thermo A-31851 IF-P/IF-F/ICC Alexa-555 Fisher (1:350) ScientificMMP1 Gt Poly, IgG Sigma- M4696- 10 μg/mL nonconjugated Millipore 100UGMatrix MMP 2 Ms Mono. IgG Abcam ab86607 IHC-P (1:300) Metallo- (C-[6E3F8] proteinases terminal)

TABLE 4 Secondary Antibodies Fluorogenic Anti-Gt- Dk Poly, DyLight IgGThermo SA5-10086 1:1000 assay 488 488 (H + L) Fisher (Cross ScientificAdsorbed) Anti-Rb- Dk Poly, DyLight IgG Thermo SA5-10040 1:1000 594 594(H + L) Fisher Scientific Anti-Ms- Dk Poly, DyLight IgG Thermo SA5-101661:1000 488 488 (H + L) Fisher Scientific Anti-Gt- Dk Poly, DyLight IgGThermo SA5-10085 1:1000 350 350 (H + L) Fisher Scientific Anti-Rb- DkPoly, Alexa IgG Thermo A21206 1:400 488 Fluor 488 (H + L) FisherScientific Anti-Gp- Dk Poly, Alexa IgG Jackson 706-605- 1:400 647 Fluor647 (H + L) Immuno 148 Research Anti-Ms- Dk Poly, Alexa IgG ThermoA21207 1:400 594 Fluor 594 (H + L) Fisher Scientific Chromogenic Anti-RbHs Micropolymer IgG Vector MP-7401 Ready-to-use assay Detection HRPlaboratories Kit Anti-Ms Hs Micropolymer IgG Vector MP-7402 Ready-to-useDetection HRP laboratories Kit Anti-Gt Hs Micropolymer IgG VectorMP-7405 Ready-to-use Detection HRP laboratories Kit Anti-Ms HsMicropolymer IgG Vector MP-7402 Ready-to-use Detection HRP laboratoriesKit

Abbreviations

-   -   Host: Gt, goat; Rb, rabbit; Dk, donkey; Hs, horse; Ms, mouse;        Gp, Guinea Pig    -   Clonality or Conjugation: Poly, polyclonal Mono-C #, monoclonal        clone number    -   Application: IHC, immunohistochemistry IHC-F,        immunohistochemistry-frozen sections IHC-P,        immunohistochemistry-paraffin embedded samples ICC,        immunocytochemistry IF, immunofluorescence HHP, horseradish        peroxidase

Samples were rinsed twice with PBS for 5 min, incubated for 20 min atroom temperature with secondary biotinylated antibody (LSAB+ System-HRP,code K0690; Dako, Glostrup, Denmark) and then with Streptavidin-HRP(LSAB+ System-HRP, code K0690, Dako, Glostrup, Denmark).Diaminobenzidine (Dako, Glostrup, Denmark) was used as substrate, andsections were counterstained with hematoxylin (PMID: 29248458). Forimmunofluorescence, non-specific protein binding was blocked by 5%normal goat serum. Specimens were incubated overnight at 4° C. withprimary antibodies (chicken anti-GFP, Abcam, code: ab13970,dilution=1:200; rabbit anti-HNF4alpha, Abcam, code: 92378, dilution:1:50, rabbit anti-albumin, ab2406, dilution=1:500). Specimens werewashed and incubated for 1 h with labeled isotype-specific secondaryantibodies (anti-chicken AlexaFluor-546, anti-mouse Alexafluor-488,anti-rabbit Alexafluor-488, Invitrogen, Life Technologies Ltd, Paisley,UK) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) forvisualization of cell nuclei (PMID: 26610370). For all immunoreactions,negative controls (the primary antibody was replaced with pre-immuneserum) were also included. Sections were examined in a coded fashion byLeica Microsystems DM 4500 B Light and Fluorescence Microscopy (LeicaMicrosystems, Weltzlar, Germany), equipped with a Jenoptik Prog Res C10Plus Videocam (Jena, Germany). Immunofluorescence stains were alsoanalyzed by Confocal Microscopy (Leica TCS-SP2). Slides were furtherprocessed with an Image Analysis System (IAS—Delta Sistemi, Roma-Italy)and were independently evaluated by two researchers in a blind fashion.Immunofluorescence stains were scanned by a digital scanner (AperioScanscope FL System, Aperio Technologies, Inc, Oxford, UK) and processedby ImageScope.

Quantitative Reverse Transcription and Polymerase Chain Reaction(qRT-PCR).

Total RNA was extracted from the organoids or grafts using Trizol(Invitrogen). First-strand cDNA synthesized using the Primescript 1ststrand cDNA synthesis kit (Takara) was used as a template for PCRamplification. Quantitative analyses of mRNA levels were performed usingFaststart Universal Probe Master (Roche Diagnostics) with ABI PRISM7900HT Sequence Detection System (Applied Biosystems). Primers weredesigned with the Universal Probe Library Assay Design Center (RocheApplied Science). Primer sequences are listed in Table 5, below. Theprimers were annealed at 50° C. for 2 min and 95° C. for 10 min,followed by 40 cycles of 95° C. (15 s) and 60° C. (1 min). Expression ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used generally as acontrol and a standard.

TABLE 5 Primers (qPCR) Accession Length Tm Category Name NumberSequence (5′ to 3′) (bp) (° C.) Housekeep- GAPDH NM_001206359.1 Sense:473 60° C. ing gene ATCCTGGGCTACACTGAGGAC (SEQ ID NO: 1) Anti-sense:AAGTGGTCGTTGAGGGCAATG (SEQ ID NO: 2) Pluripotency Nanog NM_001129971.1Sense: 214 62° C. genes TTCCTTCCTCCATGGATCTG (SEQ ID NO: 3) Anti-sense:ATCTGCTGGAGGCTGAGGTA (SEQ ID NO: 4) Sox2 NM_001123197 Sense: 216 60° C.GCCCTGCAGTACAACTCCAT (SEQ ID NO: 5) Anti-sense: GCTGATCATGTCCCGTAGGT(SEQ ID NO: 6) Oct4 JN633978.1 Sense: 176 60° C. CGAAGCTGGACAAGGAGAAG(SEQ ID NO: 7) Anti-sense: GCTGAACACCTTCCCAAAGA (SEQ ID NO: 8)Endodermal EpCAM NM_214419.1 Sense: 314 53° C. genesACCAGAGAATGCTATCCAGAAC (SEQ ID NO: 9) Anti-sense: CTCACTCGCTCCAAACAGG(SEQ ID NO: 10) Lgr5 NM_001315762.1 Sense: 110 60° C.CCTTGGCCCTGAACAAAATA (SEQ ID NO: 11) Anti-sense: ATTTCTTTCCCAGGGAGTGG(SEQ ID NO: 12) Sox9 NM_213843.2 Sense: 229 60° C. CGGTTCGAGCAAGAATAAGC(SEQ ID NO: 13) Anti-sense: GTAATCCGGGTGGTCCTTCT (SEQ ID NO: 14) Bmi1NM_001285971.1 Sense: 189 60° C. TCATTGATGCCACAACCATT (SEQ ID NO: 15)Anti-sense: TGAAAAGCCCCGGAACTAAT (SEQ ID NO: 16) GI tract- Muc2NC_010444.3 Sense: 249 60° C. related GGCTGCTCATTGAGAGGAGT genes(SEQ ID NO: 17) Anti-sense: ATGTTCCCGAACTCCAAGG (SEQ ID NO: 18) CDX2NC_010453.4 Sense: 115 56° C. AGAACCCCCAGGTCTCTGTCTT (SEQ ID NO: 19)Anti-sense: CAGTCCGAAACACTCCCTCACA (SEQ ID NO: 20) Hepatic AFPNM_214317.1 Sense: 609 60° C. parenchymal CGCGTTTCTGGTTGCTTACACcells related (SEQ ID NO: 21) genes Anti-sense: ACTTCTTGCTCTTGGCCTTGG(SEQ ID NO: 22) Albumin AY663543.1 Sense: 115 56° C.AGTCTGCCAAGCTGCTGATA (SEQ ID NO: 23) Anti-sense: AGCCTTGGGAAATCTCTGGC(SEQ ID NO: 24) Pancreatic PDX-1 NM_001141984.2 Sense: 139 62° C.endocrine- AGTGATACTGGATTGGCGTTG related (SEQ ID NO: 25) genesAnti-sense: TAGGGAGCCTTCCAATGTGT (SEQ ID NO: 26) MAFA NC_010446.4 Sense:120 62° C. GCTTCAGCAAGGAGGAGGTC (SEQ ID NO: 27) Anti-sense:TCTCGCTCTCCAGAATGTGC (SEQ ID NO: 28)

RNA-sequencing and Gene Expression Analyses. RNA was purified from cellsusing the Qiagen Rneasy Kit. RNA integrity (RIN) analysis was performedusing an Agilent 2000 Bioanalyzer. The cDNA libraries were generatedusing the Illumina TruSeq Stranded mRNA preparation kit and sequenced onthe Illumina HiSeq 2500 platform. Two samples were sequenced per lane,occupying a total of 8 lanes for all of the samples (one flow cell).Quality control analysis was completed using FastQ. Mapping of sequencereads to the human genome (hg19) was performed with MapSplice2 usingdefault parameters. Transcript quantification was carried out by RSEManalysis, and DESeq was used to normalize gene expression and identifydifferentially expressed genes. MapSplice2 was also used to detectcandidate fusion transcripts. Fusion calls were based on the depth andcomplexity of reads spanning candidate fusion junctions. Gene expressionprofiles were compared using Pearson's correlation analysis andhierarchical clustering was performed in R. Hierarchical clustering wasperformed following Variance Stabilizing Transformation provided in theDESeq package. Pathway enrichment analysis was performed with theIngenuity Pathway Analysis (IPA) software. Differential gene expressionanalysis was conducted only on genes with a minimum average normalizedcount >50 in at least one category.

Statistical Analyses.

Statistically significant differences between samples were calculated byusing Student's 2-tailed t test and results are presented as themean±standard deviation (SD). P values of less than 0.05 were consideredstatistically significant.

Example 2: Administration of a Patch Graft to the Livers of Pigs

Representative findings from studies in which organoids of porcine GFP+BTSCs/ELSMCs were transplanted onto the livers of wild type pigs. Thefindings are presented in FIGS. 3-4, 6, 8-10, 14). The cells were ableto engraft within a week or less and caused remodeling of the GlissonCapsule and the subjacent tissue during the engraftment process (FIGS.3-4, 6, 8-10). The remodeling process demonstrated parallels withinflammatory processes (FIGS. 9A-9B). By week 2-3, the engrafted cellsdemonstrated maturation to adult cell types comprising hepatocytes andcholangiocytes (FIGS. 4A-4E). The engraftment process proved mediated bymultiple matrix metalloproteinases (FIGS. 6A-6H) that caused thedramatic remodeling of the host tissue. With clearance of the graftbiomaterials, the domain of paracrine signals (matrix and solublesignals) resulted in muting of the MMPs followed by maturation of thedonor cells to adult cell types. By three weeks, the host tissue hadstabilized and exhibited fully integrated donor and host cellsthroughout. Proof of the distribution of the donor cells into allregions of the host liver was achieved with PCR analyses for DNAencoding GFP (FIG. 14). The patch grafting method proved safe for thehost and for the tissue. The only difficulties proved to be skininfections at the surgery site exacerbated by immuno suppression of thepigs (FIGS. 12A-12E).

Example 3: Example 3: Administration of a Patch Graft to the Livers ofMice

Patch grafts containing organoids of porcine GFP+ BTSCs/ELSMCs wereattached surgically to the livers of FAH/NRG mice. The livers wereevaluated a month after transplantation. Donor cells were foundthroughout the host livers (FIGS. 3C, 5A-5P, 11, 13A-13C). As for thepatch grafts on pig livers, those on the mouse livers resulted in rapidengraftment and integration throughout the host liver (FIGS. 13A-13C).Indeed, by a month, the donor cells largely took over the host liver.Ongoing studies are assessing the extent of domination by donor versushost cells at varying time points following surgical attachments of thegrafts.

Example 4: Administration of a Patch Graft to the Pancreas

This example describes an exemplary method for administering a patchgraft to an organ, such as a pancreas. Specifically, the detailedprotocols describe the administering a patch graft to the pancreas of amutant mouse (Akita/NRG) that is immunocompromised and also has agenetic condition that makes it prone to diabetes.

Preparation of Work Area.

Surgical tools are autoclaved prior to surgery. The entire surgicalprocedure is conducted in a laminar flow cabinet to minimizeenvironmental contaminants. The necessary supplies are assembled on thepreparation, surgical and recovery area, using proper aseptic technique.A recirculating water heating pad is prepared. The recirculating waterheating pad has a temperature of 38° C. and is used for temperaturestabilization during surgery. The heating pad is covered with a sterilewaterproof pad. An operating microscope, such as UB56, which is providedby UNC Animal Facility, is used during the surgical procedure. Aninstrument sterilizer, such as a hot bead sterilizer, is used tosterilize instruments in between surgical procedures. Lastly, a recoveryarea consisting of a clean cage, lined by flat paper bedding, isprepared.

Preparing the Animals for Surgery.

For patch graft surgery, 8-week old male mice were used. The 8-week oldmale mice were housed in standard cages and maintained on a 12-hourlight/12 hour dark cycle and fed a standard rodent diet ad libitum. Themice were anesthetized by peritoneal injection of 100 mg/kg of ketamineand 10 mg/kg of xylazine. Proper anesthetization was assessed byobserving gradual loss of voluntary movement and muscle relaxation. Theloss of reflexes was tested by toe pinching. Ophthalmic ointment wasapplied to prevent dryness of the eyes while under anesthesia.

Surgical Site Preparation.

The thorax and abdomen were disinfected with antiseptic chlorhexidinesolution. Fur was removed from the area of the abdomen (approximately2.5 cm×1.5 cm) by shaving. The shaven area is disinfected using gauzesoaked with chlorhexidine solution, followed by an alcohol solution, anda final application of chlorhexidine solution. The animal is positionedin the surgical area such that the prepared surgical site is facingupwards toward the surgeon. To create a sterile working field, awaterproof surgical drape is draped over the mouse such that thedisinfected abdominal region is exposed while the rest of the body iscovered. Prior to performing the procedure, the mouse was monitored fordepth of anesthesia.

Secure Patch on Surface of Pancreas.

An upper midline incision is made in the skin extending from the xiphoidprocess to the umbilicus using a sterile surgical blade. The underlyinglinea alba and the peritoneum are separated using sterile scissors inorder to expose the upper abdominal quadrant. To prevent drying-out ofthe internal organs, the internal organs are regular sprinkled with asterile solution of 0.9% sodium chloride. Using sterile tweezers orswab, the stomach is retracted superiorly, exposing the spleen and thesplenic lobe (the tail region) of the pancreas. A patch graft (see belowfor its preparation) soaked in fresh, serum-free Kubota's medium in aculture dish is placed on the surface of the pancreas. The patch cornersare secured with Surgical Adhesive (Patterson Veterinary, Devens,Mass.).

Preparation of the Patch Graft.

The backing for the patch graft is prepared well in advance of thesurgery. The patch graft containing the cells in a soft hydrogel and ontop of the backing is prepared a few hours before the surgery. The cellsand the hyaluronan mixtures yielding hydrogels are all prepared inserum-free Kubota's Medium. There are three distinct hyaluronanhydrogels used varying in level of viscoelasticity (rigidity), withrigidity levels of 1×, 2×, and 10×, achieved by varying the ratio of theconcentration of the thiol-modified hyaluronan with that of thepolyethylene glycol diacrylate (PEGDA). The most rigid (˜600-800 Pa) ofthe 3 hydrogels (the 10×) is used to impregnate the backing of silk andis prepared well in advance (e.g. the day before) of the surgicalprocedures. At the time of the surgery, the soft hydrogel is prepared bymixing hyaluronan with PEGDA at a 4:1 ratio (50 μl of hyaluronan ofwhich 20% is PEGDA) and is allowed to gel for about 5 minutes. Then theorganoids (1×10⁵) are mixed into this soft hydrogel; the mixture layeredonto the backing; and the hydrogel/cell mixture allowed to continue thegelation process for another 30 minutes to 2 hours. The patch graft cannow be tethered to the target site. For most tissues and organs, thiscan be by means of sutures at the corners of the grafts. For sensitivetissues such as the pancreas, it can be with surgical glue or, if thepatch graft is positioned in part over duodenum and in part overpancreas, then use of sutures for the duodenum and surgical gue for thepancreas. After attachment of the graft, the patch grafts are coatedwith 200 μl of a HA hydrogel (viscoelasticity=200-300 Pa) to minimizeadhesions by nearby tissues and organs.

The organs are placed back into the abdominal cavity. The incision isclosed using 3.0 polyglycol filamentous thread in a continuous suturepattern for the muscle/peritoneal layer and in a discontinuous suturepattern for the skin.

Post-Operational Care and Monitoring.

After the surgical procedure is complete, the mouse is placed in therecovery area, which consists of a cage placed on a heating pad andlined with flat paper bedding in order to maintain normal bodytemperature. To avoid post-operative hypoglycemia, nutritional support(DietGel Recovery, ordered from ClearH₂O company) is provided by placingmoistened food on the bottom of the cage. Moistened food is prepared bysoaking standard rodent diet pellets in water until they soften. Fluidsupport is provided by the moistened food and administration of water adlibitum. Buprenophine is used as analgesia (0.05-0.1 mg/kg) twice dailyfor 3 days post-surgery. During the entire experimental follow-up, themice are observed for occurrence of possible signs of infection,including secretion of liquid or pus from the wound, or for physicaldeterioration characterized by reduction in grooming behavior andactivity level, lower appetite and bodyweight loss.

Example 5: Engrafted BTSCs/ELSMCs Rapidly Reverse Hyperglycemia inDiabetic NRC/Akita Mice

In this example the use of patch grafts for the treatment of diabeteswas investigated.

Mouse Models.

Two mouse rr odels were used in this example, Akita mice and theNRG/Akita mice. The Akita mice (DBA-Akita Inst Akita) on the DBAbackground is a recently described mouse mutant model of type I diabetesmellitus. Diabetes is the result of selective pancreatic β-cell toxicityand depletion resulting from misfolding of insulin2. Mice heterozygousfor the Akita spontaneous mutation (Ins2^(Akita)) are viable andfertile. They have hyperglycemia, hypoinsulinemia, polydipsia andpolyuria beginning around 3-4 weeks of age. Studies carried out by theAnimal Models of Diabetic Complications Consortium (AMDCC) indicatedthat the DBA/2J strain is especially prone to developing diabetic,nephropathic complications. Therefore, the advantage of this model oftype I diabetes is the early onset progressive, functional andstructural glomerular injury, and the fact that the diabetes occursspontaneously.

The NRG/Akita mice are immunocompromised mice that have been geneticallyaltered to have the genetic defect of the Akita mice. Breeding pairs ofthe two mouse models were ordered from the Jackson Laboratory (BarHarbor, Me.) and used to establish colonies at UNC. Eight-week-old micewere used for patch grafting studies that lasted for approximately onemonth after which the mice are euthanized.

Patch Graft Administration and Results.

Patch grafts were produced as described in Example 1. Patch graftsurgery, in which patch grafts containing cells were administered to thepancreas of mice as described in Example 4. In controls, surgery wasalso performed with graft biomaterials but without cells (e.g., micetreated with graft biomaterials only).

After the surgery, blood glucose levels were obtained from mice treatedwith patch grafts, mice treated with graft biomaterial only, anduntreated mice (e.g., mice that did not receive patch grafts nor graftbiomaterials). Mice treated with patch grafts showed a progressivereduction of blood glucose levels compared with mice treated withbiomaterial only and untreated mice. The levels reduced were significantby 1 week after patch grafting and the effects were sustained for up to3-4 weeks after which the mice were euthanized. Blood glucose levelswere dynamically monitored using a glucose meter every other daystarting 3 days after surgery in animals, each one after a 5-hour-fast.The results showed that hyperglycemia was alleviated in diabetic micetreated with patch grafts by around 7-10 days, and a few mice werecorrected to euglycemia within 3 weeks (FIG. 15A, triangle). No suchalleviation in hyperglycemia was observed in untreated diabetic mice(FIG. 15A, circle) nor in diabetic mice treated with biomaterial only(FIG. 15A, square). In addition, the gradual drop in glycemia was foundto correlate with significantly increasing serum levels of C-peptide,which is indicative of insulin secretion (FIG. 15B, see third column atD7, D14, and D21, which corresponds to diabetic mice treated with patchgrafts). Murine serum C-peptide levels were minimal in untreateddiabetic mice (FIG. 15B, first column under D0, D7, D14, and D21),diabetic mice treated with biomaterial only (FIG. 15B, second columnunder D0, D7, D14, and D21), and diabetic mice treated with patch graftsat day 0 (FIG. 15B, third column under D0).

Serum insulin levels also gradually increased in diabetic mice treatedwith patch grafts FIG. 15C, third column under D21 and D28). Seruminsulin levels did not increase in untreated diabetic mice (FIG. 15C,first column under D21 and D28) or diabetic mice treated with biomaterial only (FIG. 15C, second column under D21 and D28).

Intraperitoneal glucose tolerance tests (IPGTT) were also performed toassess the in supra-physiological glucose-stimulated insulin release.The measurement of this glucose spike was subsequently assessed over aperiod of time to determine the response of insulin to increased plasmaglucose. Compared with non-diabetic mice (normal mouse controls, FIG.15D, triangle), diabetic mice treated with biomaterial only (FIG. 15D,circle) showed a high basal glycemia and an altered glucose tolerancetest with sustained hyperglycemia. By contrast, the diabetic micetreated with patch grafts containing cells (FIG. 15D, square) showednormal (or near normal) basal blood glucose levels and a response toIPGTT comparable to non-diabetic control mice (FIG. 15D, triangle). Thedata provides additional evidence for how the patch grafts enabled theseformerly diabetic mice with the ability to tolerate and manage glucosespikes. The complete return to euglycemia in organoid-transplanted micewithin 240 minutes is striking evidence of the remarkable ability of theBTSC cells to respond to supra-physiological glucose spikes. However,the correction of hyperglycemia was significantly slower than thecorrection observed in nondiabetic control mice.

In FIGS. 16A-16B-18A-18B are given images of pancreas sections fromAkita/NRG mice with patch grafts from DS-red mice. The sections werestained for insulin (FIGS. 16A-16B) or NGN3 (FIGS. 17A-17C and 18A-18B).Donor cells are depicted red in FIGS. 18A-18B as visualized by DS redstaining.

Example 6. Patch Grafts Containing Organoids of Porcine GFP+BTSCs/ELSMCs Surgically Attached to the Pancreas of Wild Type Piglets

In this example, the grafting of patch grafts comprising GFP+BTSCs/ELSMCs organoids from a transgenic pig was done onto the pancreasof a wild type pig.

Patch grafts containing organoids of porcine GFP+ BTSCs/ELSMCs weretethered onto the pancreas of wild type piglets and evaluated after oneweek. The GFP+ donor cells engrafted within a week and were found widelydispersed throughout the pancreas (FIGS. 19A-19B-21A-21C). At the firstweek time point, the engrafted cells had matured into both islets andacinar cells. However, the nuclear biomarker, GFP, was observed in thenucleus of the islets but in the cytoplasm of the acinar cells. Studiesare ongoing to assess if GFP stabilizes to the nucleus at later timepoints, as occurred with engraftment in the liver.

In FIGS. 19A-19B-21A-21C, the green cells, whether islets or acinarcells, are the donor cells. Sections were also stained for insulin (red)such that those that are red with blue nuclei are host islets, whereasthose that have red/purplish nuclei and yellowish cytoplasm are donorcell-derived beta cells in the islets. The antibodies used to detectinsulin, glucagon, GFP, and amylase are shown in the table below:

Fluoro- Host Species Species Antibody chrome and Isotype ReactivityManufacturer Catalog # Anti-Insulin Guinea Pig Human, Pig Abcam ab195956Polyclonal Anti- Mouse Human, Pig, Sigma-Aldrich G2654 GlucagonMonoclonal Rat, Guinea IgG1 Pig, Feline, Mouse, Canine, Rabbit Anti-GFPRabbit Aequorea Novus NB600-308 Polyclonal victoria Biologicals Anti-Mouse Human, Santa Cruz sc-46657 Amylase 2B Monoclonal Mouse, RatBiotechnology IgG2A Anti Guinea Alexa Donkey ImmunoResearch 706-605-148Pig IgG Fluor 647 Laboratories Anti Mouse Alexa Donkey ThermoFisherA-21203 IgG Fluor 594 Scientific - Invitrogen Anti Rabbit Alexa DonkeyThermoFisher A-21206 IgG Fluor 488 Scientific - Invitrogen

Immunofluorescent staining of the porcine pancreas that has been graftedwith GFP+ BTSCs/ELSMCs from transgenic pigs are shown in FIGS. 19A-19B.Immunofluorescent staining for insulin is shown as red and GFP as green.The region of the graft was harvested and analyzed at day 7 posttransplantation. Nuclei were stained with DAPI(4′,6-Diamidine-2′-phenylindole dihydrochloride) and appear blue. TheGFP+ cells were inherently green from the transgene linked to the H2Bhistone but the intensity of the green color was enhanced by stainingwith an antibody to GFP and that was coupled to a green fluoroprobe.Large numbers of GFP+ donor-derived cells are visible in the proximityof the area where the patch graft was placed. Insulin expression,characteristic of pancreatic islet beta cells, was identified with ananti-insulin antibody coupled to a red fluoroprobe. Endogenous (host)islet beta cells appear red and are evident in the upper portion of thepancreas. Donor-derived beta cells appear with yellowish nuclei from themerge of the blue (DAPI) and green (GFP) and a cytoplasm that isred/orange from staining for insulin in the lower portion of thepancreas, proximal to the site of placement of the patch graft. This lowmagnification image demonstrates the extent of engraftment into thepancreas, as well as the engraftment into the submucosal region of theduodenum, the location of Brunner's Glands (hypothesized to be thestarting point of the network of cells contributing to organogenesis ofthe liver and pancreas).

Immunofluorescent staining for amylase and GFP in a sequential sectionfrom the same tissue block is shown in FIG. 19B. Amylase (green) isdetected predominantly in pancreatic acinar tissue, as well as in themucosal layer and in the lumen of the duodenum. Insulin (red) does notoverlap with Amylase (green). This staining, in combination with that ofFIG. 19A, suggests that a large portion of GFP+ donor-derived cellscommitted to a pancreatic acinar-like fate.

Immunofluorescent staining for insulin and GFP in pancreatic tissuesections from 3 recipients of GFP+ BTSCs/ELSMCs patch grafttransplantation (day 7 post transplantation) is shown in FIGS. 20A-20C.GFP+ cells were observed in the parenchyma of the pancreas, in theproximity of the patch graft site, in all recipients. Notably, GFP+cells appeared at several millimiters of distance from the patchmaterial, and appeared to be well integrated into the parenchyma of therecipient pancreas. Patch material (SERI silk) presented a degree offluorescence in different channels and is still visible at day 7 posttransplantation.

FIGS. 21A-20C demonstrate the co-existence of endogenous (host) isletbeta cells (Insulin+/GFP−: red cytoplasm) and donor-derived islets betacells (Insulin+/GFP+: yellow-orange cytoplasm) in the pancreas at 7 dayspost transplantation of GFP+ BTSCs/ELSMCs patch graft. Donor-derivedislet beta cells and endogenous (host) islet beta cells were observed inall cases. The majority of GFP+ cells presented a phenotype consistentwith that of pancreatic acinar cells. GFP+ cells organized to form aductal structure are visible in the lower portion of FIG. 21A.

Example 7. Use of Patch Grafts of BTSCs/ELSMCs to Treat Diabetes Inducedin Animals by Streptozocin (STZ)

This example describes the detailed protocol for an exemplary method fortreating diabetes by administering patch grafts to mice made diabetic bytreatment with STZ.

Animal Care and Housing.

8-14-week-old C57BL/6 (Jax Stock 000664), BALB/c (Jax stock 000651), andDsRed.MST B6 (Jax Stock 006051) are housed in standard cages, maintainedon a 12 hour light/12 hour dark cycle, and fed a standard rodent diet adlibitum.

8-14-week-old immunodeficient NSG mice (Jax Stock 005557) are housed ina germ-free environment on a 12 hour light/12 hour dark cycle and fed anautoclaved diet ad libitum.

DSRed.MST B6 mice are used as donors of islets and BTSCs/ELMCs.

C57BL/6, BALB/c, and NSG mice are used as recipients.

Day −14 Diabetes Induction.

Diabetes is induced in prospective recipients via streptozotocintreatment at a dose of 250 mg/kg. After the first injection, glycemicvalues are monitored and an animal is considered diabetic if glycemia isfound to be >350 mg/dl for 3 consecutive days. In rare cases the firstdose of streptozotocin may result only partially effective (e.g.,glycemic values not reaching >350 mg/dl for 3 consecutive days), hencesuch animals receive additional dosing(s) to complete beta celldepletion and induction of sustained hyperglycemia. Streptozotocintreatment is repeated for a maximum of 3 times, with each injection 3days apart. 95-100% of the animals treated with streptozotocin areexpected to develop diabetes.

Diabetic animals are maintained with LP insulin pellets, placed in thesubdermal space. Insulin pellets will be removed at +15 days after patchgraft transplantation.

Day 1:

Hyaluronic Acid (HA) hydrogel is combined with 20% polyethylene glycoldiacrylate (PEGDA) linker solution. After the soft HA gel constituentsare mixed, they are allowed to gel for ˜5 minutes and then are added theBTSCs/ELMCs organoids (1×10⁵) or islets (2000 IEQ) and the gelationallowed to continue for another 30 minutes to 3 hours. This is added ontop of a premade backing containing the more rigid hyaluronan layer;this was prepared a day ahead of time and allowed to complete gelatinovernight by culturing in a 37° C. 5% CO₂ incubator at the air-liquidinterface.

Preparation of Work Area.

Autoclave surgical tools prior to surgery. Appropriate protectivecovering is worn for the surgery. Multiple sets of sterile gloves areavailable for use in the surgical work area. The entire surgicalprocedure is performed in a laminar flow cabinet to minimizeenvironmental contaminants. Necessary supplies are assembled on thepreparation, surgical and recovery area, using proper aseptic technique.A heating pad at a temperature of 38° C. is prepared for temperaturestabilization during surgery. Surgery is performed with the use of anoperating microscope. An instrument sterilizer, such as a hot beadsterilizer, is used to sterilize instruments in between surgicalprocedures. A recovery area consisting of a clean cage, lined by flatpaper bedding is prepared.

Preparation of the Animals for Surgery.

A set of transplantations are sex-mismatched: cells from male donorswill be transplanted in female recipients, to enable Y chromosometracing and confirmation of fluorescent tracing.

-   -   For Patch Graft transplantation of DsRed.MST B6 cells in the        syngeneic setting, use 8-14 week old C57BL/6 recipient mice.    -   For Patch Graft transplantation of DsRed.MST B6 cells in the        allogeneic setting, use 8-14 week old BALB/c recipient mice.    -   For Patch Graft transplantation of human cells, use 8-14 week        old immunodeficient NSG recipient mice.

The mouse is weighed and the required dose of Ketamine (100 mg/kg) andXylazine (10 mg/kg) is calculated. The mouse is anesthetized byintraperitoneal injection of 100 mg/kg of Ketamine and 10 mg/kg ofXylazine. Proper anesthetization is assessed by observing gradual lossof voluntary movement and muscle relaxation. The loss of reflexes istested by toe pinching. An ophthalmic ointment is applied to preventdryness of the eyes while under anesthesia. As an alternative,isoflurane 2% via inhalation may be used to anesthetize the mouse.

Surgical Site Preparation.

Fur is removed by shaving from the abdomen (approximately an area of 2.5cm×1.5 cm). The thorax and abdomen are disinfected with antisepticchlorhexidine solution. The shaved area is disinfected using gauzesoaked with chlorhexidine solution, then wiped with alcohol solution,and applying again the chlorhexidine solution. The animal is positionedand secured in the surgical area, by laying the animal on its back suchthat the prepared surgical site in the abdominal region is upwardsfacing the surgeon. The mouse is draped using a waterproof surgicaldrape with an opening that leaves the disinfected abdominal regionexposed while covering the rest of the body to create a sterile workingfield. The mouse is monitored prior to the procedure for depth ofanesthesia.

Secure Patch on Surface of Pancreas.

An upper midline incision is made in the skin extending from the xiphoidprocess to the umbilicus using a sterile surgical blade. The incision inthe skin is extended from the xiphoid process towards the upper limbs, 1cm in each direction. The incision is extended from the umbilicus inboth directions toward the lower limbs, 1 cm in each direction. Theunderlying linea alba and the peritoneum are separated using sterilescissors or scalpel in order to expose the upper abdominal quadrant. Theincision is divaricated with retainer clips. To prevent drying-out ofthe internal organs, the internal organs are regularly sprinkled withsterile 0.9% sodium chloride. Sterile tweezers or swab are used toretract the stomach superiorly, and the intestine inferiorly, exposingthe spleen and the splenic lobe (the tail region) of the pancreas. Apiece of patch loaded with cells embedded in HA gel is soaked in freshKubota's Medium in a culture dish. The gel side is oriented such that itfaces the pancreas (this patch resembles a BandAid, the gel is only onone side). The patch is laid on the surface of the body of the pancreas,such that the gel side contacts the pancreas. The two patch corners aresecured with Surgical Adhesive (Patterson Veterinary, Devens, Mass.).The patch is covered with 200 μl of 2×HA hydrogel to minimize adhesionsamong organs. The organs are placed back into the abdominal cavity. Theincision is closed using 3.0 polyglycol filamentous thread in acontinuous suture pattern for the muscle/peritoneal layer and in adiscontinuous suture pattern for the skin.

Post-operational Care and Monitoring.

After the surgical procedure is completed, the mouse is placed in therecovery area. The recovery area consists of a cage placed on a heatingpad and lined with flat paper bedding in order to maintain normal bodytemperature. To avoid post-operative hypoglycemia, nutritional support(DietGel Recovery, ordered from ClearH₂O company) is provided by soakingrodent diet pellets in water until they soften and placing the moistenedfood on the cage bottom. Fluid support is provided via the moistenedfood and provide water ad libitum. Buprenophine is used as analgesia(0.05-0.1 mg/kg) twice daily for 3 days post-surgery or the SR versionwhich is 1 injection with 72 hr effect. During the entire experimentalfollow up, the mice are observed for occurrence of possible signs ofinfection, including secretion of liquid or pus from the wound, or forphysical deterioration characterized by reduction in grooming behaviorand activity level, lower appetite and body weight loss. A preventativeantibiotic, such as Clavamox or Baytril, is placed in the drinking waterfor up to 14 days post op.

What is claimed is:
 1. A method of engrafting cells into a solid organof a subject in need thereof, comprising: contacting a patch graft ontoa solid organ, the patch comprising a mixture of epithelial cells andmesenchymal cells incorporated into a biomaterial having a firstviscoelasticity property, in which the biomaterial promotes anengraftment of at least a portion of said epithelial cells, mesenchymalcells, or both among the cells of the solid organ; demonstrating that atleast a portion of said epithelial cells, mesenchymal cells, or bothhave engrafted among the cells of the solid organ.
 2. The method ofclaim 1 in which said demonstrating comprises measuring a level of asecretion from the solid organ, or a metabolic effect of the solidorgan, in a biological sample obtained from the subject to demonstratethat at least a portion of said epithelial cells have engrafted amongthe cells of the solid organ.
 3. A method of engrafting cells into asolid organ of a subject in need thereof, comprising: contacting a patchgraft onto a solid organ, the patch comprising a mixture of epithelialcells and mesenchymal cells incorporated into a hydrogel layer having afirst viscoelasticity property, in which the hydrogel promotes amigration of at least a portion of said epithelial cells, mesenchymalcells, or both from the patch through an outer surface of the solidorgan, demonstrating that at least a portion of said epithelial cells,mesenchymal cells, or both have migrated through an outer surface of thesolid organ.
 4. The method of claim 3 in which said demonstratingcomprises measuring a parameter or a change in same, which indicates aphysiological effect in the subject resulting from the migrated cells.5. The method of claim 1 in which the patch graft further comprises abacking that promotes a migration of at least a portion of the mixtureof epithelial cells and mesenchymal cells towards the solid organ. 6.The method of claim 5 in which at least a portion of the mixture ofepithelial cells and mesenchymal cells migrates over the substantialwidth of the solid organ and distributes throughout the solid organ. 7.The method of claim 1 in which the solid organ is an endodermal organ.8. The method of claim 1 in which the solid organ is an endodermal organcomprising liver, pancreas, intestine, lung, bile duct, thymus, thyroid,parathyroid and the urogenital sinus region of the prostate and vagina.9. The method of claim 8 in which the endodermal organ comprises liver,and engraftment involves a remodeling of Glisson's Capsules.
 10. Themethod of claim 8, which further gives rise to a combination of (i)engrafted epithelial cells and mesenchymal cells and (ii) host cells.11. The method of claim 8, which gives rise to functional hepaticparenchymal cells.
 12. The method of claim 11, in which the parenchymalcells comprise hepatocytes and cholangiocytes.
 13. The method of claim8, in which the endodermal organ comprises pancreas, and engraftmentinvolves a remodeling of pancreatic capsules and pancreatic tissue nearto the graft site.
 14. The method of claim 13, which gives rise tofunctional pancreatic cells.
 15. The method of claim 14, in which thefunctional pancreatic cells comprise acinar cells and islets.
 16. Themethod of claim 14, in which the pancreas secretes increased levels ofat least one of insulin, c-peptide, glucagon, somatostatin, orpancreatic polypeptide.
 17. The method of claim 14, in which thepancreas exhibits a metabolic effect of reduced blood sugar levels. 18.The method of claim 14, in which the pancreas exhibits a metaboliceffect of increased glucose tolerance.
 19. The method of claim 14, inwhich the pancreas secretes increased levels of a digestive enzyme orbicarbonate fluid.
 20. The method of claim 19, in which the digestiveenzyme comprises amylase, lipase, peptidase, ribonuclease,deoxyribonuclease, gelatinase, elastase, or combinations thereof. 21.The method of claim 14, which results in increased levels of a metabolicproduct derived from a digestive enzyme secreted by the pancreas. 22.The method of claim 21, in which the digestive enzyme comprises amylase,lipase, peptidase, ribonuclease, deoxyribonuclease, gelatinase,elastase, or combinations thereof.
 23. The method of claim 21, whichresults in improved digestion.
 24. The method of claim 11, in which theliver secretes urea, bile acids, phospholipids, lipoproteins, bilirubin,bicarbonate-rich fluids, or blood-clotting factors.
 25. The method ofclaim 4, in which a biological sample obtained from the subjectindicates reduced levels of at least one of cholesterol, blood sugar,alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactatedehydrogenase.
 26. The method of claim 1 in which the patch includes abacking positioned over the hydrogel containing of the mixture ofepithelial cells and mesenchymal cells.
 27. The method of claim 26, inwhich the backing is used to tether the hydrogel layer to the hostorgan.
 28. The method of claim 1, in which at least one of theepithelial cells, mesenchymal cells, or both are early lineage stagecells.
 29. The method of claim 28, in which the early lineage stagemesenchymal cells (ELSMCs) comprise angioblasts, precursors ofendothelia, stellate cells, or combinations thereof.
 30. The method ofclaim 28, in which the early lineage stage epithelial cells (ELSEs),ELSMCs, or both are derived from embryonic stem (ES) cells or frominduced pluripotent stem cells (iPS).
 31. The method of claim 1, inwhich the epithelial cells are mature and the mesenchymal cells areELSMCs.
 32. A method of introducing, restoring, increasing, or improvingfunctionality of a diseased, impaired, or malfunctioning solid organ ofa subject, comprising contacting the diseased, impaired, ormalfunctioning solid organ with a patch graft comprising a mixture ofepithelial cells and mesenchymal cells under conditions that promoteengraftment of the epithelial cells and mesenchymal cells; demonstratingan introduction, restoration, increase, or improvement of afunctionality in the diseased, impaired, or malfunctioning solid organ.33. The method of claim 32, in which said demonstrating comprisesmeasuring in a biological sample obtained from the subject a level of asecretion or metabolic product or effect.
 34. The method of claim 32,which further comprises demonstrating that a least a portion of themixture of epithelial cells and mesenchymal cells has distributed amongthe cells of the host organ.
 35. The method of claim 32, in which anexposed surface of the patch graft includes a coating that inhibitsadhesion of the patch graft to organs and tissues in the vicinity of thepatch graft.
 36. The method of claim 32, in which the solid organcomprises an endodermal organ.
 37. The method of claim 36, in which theendodermal organ comprises liver, pancreas, intestine, lung, bile duct,thymus, thyroid, parathyroid or the regions from the urogenital sinus ofthe prostate or vagina.
 38. The method of claim 33, in which the solidorgan comprises a pancreas and in which an increased level of thesecretion of at least one of insulin, c-peptide glucagon, somatostatin,or pancreatic polypeptide is measured.
 39. The method of claim 32, inwhich the solid organ comprises a pancreas and in which a reduced bloodsugar level is measured.
 40. The method of claim 32, in which the solidorgan comprises a pancreas and in which increased glucose tolerance isdemonstrated.
 41. The method of claim 32, in which the solid organcomprises a pancreas and in which increased levels of a digestive enzymeor bicarbonate fluid is demonstrated.
 42. The method of claim 41, inwhich the digestive enzyme comprises amylase, lipase, peptidase,ribonuclease, deoxyribonuclease, gelatinase, or elastase.
 43. The methodof claim 33, in which the solid organ comprises a pancreas and in whichincreased levels of a product from a digestive enzyme secreted by thepancreas is measured.
 44. The method of claim 43, in which the digestiveenzyme comprises amylase, lipase, peptidase, ribonuclease,deoxyribonuclease, gelatinase, or elastase.
 45. The method of claim 32,in which the solid organ comprises a pancreas and in which improveddigestion is demonstrated.
 46. The method of claim 33, in which thesolid organ comprises liver, and in which a secretion comprises urea,bile acids, phospholipids, lipoproteins, bilirubin, bicarbonate-richfluids, blood-clotting factors, or combinations thereof.
 47. The methodof claim 33, in which the solid organ comprises liver, and in which ametabolic effect is a reduced level of one or more of cholesterol, bloodsugar, alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, albumin, ammonia, gamma-glutamyltransferase, or L-lactatedehydrogenase.
 48. The method of claim 32, in which the solid organcomprises a liver, and the subject suffers from type 1 tyrosinemia. 49.The method of claim 33, in which a metabolic effect is a decrease inlevels of tyrosine or alpha-fetoprotein.
 50. A method of treating asubject diagnosed with a pathological condition attributable at least inpart to having a diseased, impaired, or malfunctioning solid organ,comprising (i) contacting the diseased, impaired, or malfunctioningsolid organ with a patch graft comprising a mixture of epithelial cellsand mesenchymal cells, (ii) allowing the epithelial cells andmesenchymal cells to migrate into and distribute among the cells of thehost solid organ, and (iii) demonstrating that a negative effect of saiddiseased, impaired, or malfunctioning solid organ has been alleviated inthe treated subject.
 51. The method of claim 50, in which saiddemonstrating comprises measuring in a biological sample obtained fromthe subject a level of a secretion or a metabolic product or effect. 52.The method of claim 50, in which the migration and distribution stepslead to an alleviation of the disease, impairment, or malfunction. 53.The method of claim 50, in which the solid organ is an endodermalorgan.888888
 54. The method of claim 53, in which the endodermal organcomprises liver, pancreas, intestine, lung, bile duct, thymus, thyroid,parathyroid, and the urogenital sinus regions of the prostate or vagina.55. The method of claim 53, in which the endodermal organ is pancreasand in which the subject suffers from diabetes.
 56. The method of claim55, in which increased levels of at least one of insulin, c-peptide,glucagon, somatostatin, or pancreatic polypeptide is measured.
 57. Themethod of claim 55, in which reduced blood sugar levels aredemonstrated.
 58. The method of claim 55, in which increased glucosetolerance is demonstrated.
 59. The method of claim 50, in which thesubject comprises a mammal.
 60. The method of claim 59, in which themammal is human.
 61. A patch graft comprising a mixture of epithelialcells and mesenchymal cells and one or more biomaterial layersincluding, at least: a) a first, inner layer for contacting a solidorgan, the first inner layer exhibiting a first viscoelastic property,incorporating a mixture of epithelial cells and mesenchymal cells,supporting an ability of the epithelial cells and the mesenchymal cellsto produce secreted matrix metallo-proteinases (MMPs) and promotingviability and immaturity of said epithelial cells and mesenchymal cells;b) optionally, a backing that confers a barrier to the cells migratingin a direction other than towards the solid organ, the backingexhibiting a second viscoelastic property; and c) optionally, a third,outer layer of a coating or material that minimizes is adhesions of thepatch graft to internal surfaces of a body cavity, including internalwalls and/or organs, in proximity to the patch graft; wherein saidviscoelastic properties are determined by measuring rheological traitsand expressed in Pascals (Pa).
 62. The patch graft of claim 61, in whichthe epithelial cells comprise early lineage stage epithelia cells(ELSEs) and the mesenchymal cells comprise early lineage stagemesenchymal cells (ELSMCs), or in which the epithelial and mesenchymalcells are of later lineage stages but are of comparable lineage stagesas each other.
 63. The patch graft of claim 62, in which the ELSMCscomprises angioblasts, precursors of endothelia, stellate cells, orcombinations thereof.
 64. The patch graft of claim 62, in which theELSEs and/or the ELSMCs are derived from embryonic stem (ES) cells orfrom induced pluripotent stem cells (iPS).
 65. The patch graft of claim61, in which the epithelial cells are of a later lineage stage and themesenchymal cells are early lineage stage mesenchymal cells (ELSMCs), orthe mesenchymal cells are of a later lineage stage and the epithelialcells are early lineage stage epithelial cells (ELSEs).
 66. The patchgraft of claim 61, in which the second viscoelastic property (expressedin Pa) has a greater value than the first viscoelastic property.
 67. Thepatch graft of claim 61, in which the one or more biomaterial layerscomprise a hydrogel, which further comprises minimally sulfated ornon-sulfated glycosaminoglycans.
 68. The patch graft of claim 67, inwhich the non-sulfated glycosaminoglycans comprise hyaluronans.
 69. Thepatch graft of claim 68, in which the hyaluronans comprise athiol-modified hyaluronan, whose gelation by disulfide bridge formationis triggered in the presence of polyethylene glycol diacrylate (PEGDA).70. The patch graft of claim 69, in which the rheological traits aredetermined, at least in part, by a starting concentration and rigidityof thiol-modified hyaluronan and PEGDA prior to gelation, a finalrigidity of hydrogel post-gelation achieved by the precise ratios of thevolumes of thiol-modified hyaluronan and PEGDA.
 71. The patch graft ofclaim 61, in which the first, inner layer exhibits a firstviscoelasticity of from about 50 Pa to about 150 Pa.
 72. The patch graftof claim 61, in which the optional backing contains a hyaluronanhydrogel layer that exhibits a viscoelasticity from about 600 to about800 Pa.
 73. The patch graft of claim 61, in which the optional third,outer layer comprises a hyaluronan hydrogel layer with viscoelasticityproperties of from about 200 to about 300 Pa.
 74. The patch graft ofclaim 61, in which the backing comprises silk.
 75. The patch graft ofclaim 74, in which the silk backing comprises a purified fibroin ofBombyx™ moth silk knitted into a scaffold, including Seri-Silk™ orContour Seri Silk™.
 76. The patch graft of claim 61, in which theepithelial cells comprise biliary tree stem cells (BTSCs) and themesenchymal cells comprise early-lineage-stage mesenchymal cells(ELSMCs).
 77. The patch graft of claim 76, in which the ELSMCs compriseangioblasts and their immediate descendants, precursors to endotheliacells, precursors to stellate cells, or combinations thereof.
 78. Thepatch graft of claim 77, in which the angioblasts express CD117, CD133,VEGFr, but do not express CD31.
 79. The patch graft of claim 77, inwhich the precursors to endothelia cells express CD133, VEGFr, CD31 andVan Willebrand Factor.
 80. The patch graft of claim 77, in which theprecursors to stellate cells express CD146, ICAM-1, alpha-smooth muscleactin (ASMA) and are negative for vitamin A.
 81. The patch graft ofclaim 61, in which the mixture of epithelial cells and mesenchymal cellsis produced by depleting cell suspensions of mature mesenchymal cells,optionally, by repeated panning procedures to remove cells that attachwithin from about 15 minutes to about 30 minutes on tissue culturedishes or surfaces at 37° C.
 82. The patch graft of claim 81, in which aculturing of the remaining cell suspensions is performed onlow-attachment dishes and in a serum-free medium until a plurality oforganoids is formed by self-assembly of epithelial cells and mesenchymalcells.
 83. The patch graft of claim 82, in which the serum-free mediumcomprises a basal medium (with no copper, low calcium (0.3 mM), 1 nMselenium, 0.1% bovine serum albumin (purified, fatty-acid-free; fractionV), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg/mltransferrin/Fe, 5 μg/ml insulin, and a mixture of purified free fattyacids that are presented complexed with fatty acid free highly purifiedalbumin.
 84. The patch graft of claim 83, in which the serum-free mediumfurther comprises 10 μg/ml high density lipoprotein.
 85. The patch graftof claim 82, in which the plurality of organoids is formed after about 2hours, after about 4 hours, after about 6 hours, after about 8 hours,after about 10 hours, after about 12 hours, after about 14 hours, afterabout 16 hours, after about 18 hours, after about 20 hours, after about22 hours, or after about 24 so hours.
 86. The patch graft of claim 82,in which the plurality of organoids comprises BTSCs positive for: a) atleast one marker selected from the group of pluripotency genesconsisting of OCT4, Sox2, Sall4, Nanog, Klf5, Cdx2 and Bmi1, b) at leastone marker selected from the group of endodermal transcription factorsconsisting of Sox9, Sox17, Pdx1, HNF4alpha, HNFB1 and ONECUT2, c) atleast one marker selected from the group of surface markers associatedwith stem/progenitors consisting of EpCAM, NCAM, LGR5, one or moreisoforms of CD44, CXCR4, sodium iodide symporter (NIS), CD49 (integrinA6), CD29 (integrin B1) and integrin B4; wherein the BTSCs are negativefor markers of mature hepatic or pancreatic cells, including P450s,aquaporin, enzymes involved in bile production, amylase and digestiveenzymes.
 87. The patch graft of claim 61, in which the one or morebiomaterial layers comprise recombinant MMPs.
 88. The patch graft ofclaim 61, in which the one or more biomaterial layers comprise cellsengineered to express MMPs.